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United States Patent |
5,516,700
|
Smith
,   et al.
|
May 14, 1996
|
Automated urinalysis method
Abstract
A method that provides techniques for determination of urinary constituents
(Blood (Red Blood Cells/Hemoglobin), Leukocytes, pH, Specific Gravity,
Bacterial Reductase/Nitrite/Indole activity, Total Ketone Bodies, Protein,
and Glucose) at low chemically significant levels with a carrier
independent reagent system that can be placed on a high throughput
autoanalyzers. Thus, giving the analyst the ability to run multiple
urinary assays on a single sample of urine simultaneously with the ability
to compare to reference standards on the same run. This system is designed
to neutralize urinary interfering substances. This method is fast,
efficient, an adaptable to many of the currently available discrete and
continuous flow automated analyzers, effective at sample to reagent ratios
of 1 to 13 or more. This method is applicable to samples with high
turbidity, high ionic strength, high color content, wide pH extremes, and
buffer strengths, among other interfering substances.
Inventors:
|
Smith; Jack V. (St. Peter, FL);
Carter; Jesse M. (Tampa, FL)
|
Assignee:
|
Chimera Research and Chemical, Inc. (Largo, FL)
|
Appl. No.:
|
429292 |
Filed:
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April 24, 1995 |
Current U.S. Class: |
436/164; 436/17; 436/63; 436/175 |
Intern'l Class: |
G01D 021/75 |
Field of Search: |
422/56-58,61
436/66,135,63,904,17-18,174-176,164
|
References Cited
U.S. Patent Documents
3907503 | Sep., 1975 | Betts et al. | 422/67.
|
4755472 | Jun., 1988 | Ismail et al. | 422/57.
|
5128265 | Feb., 1992 | Meiattini | 436/17.
|
Foreign Patent Documents |
5256853 | Mar., 1992 | JP | 436/17.
|
Primary Examiner: Alexander; Lyle A.
Attorney, Agent or Firm: Larson; Herbert W.
Parent Case Text
This application is a continuation of Ser. No. 08/068,956 filed May 28,
1993, now abandoned.
Claims
What is claimed is:
1. A method for detecting white blood cells in a patient's urine comprising
placing an aliquot of the urine to be tested in an automated analyzer
sampling cup,
placing the cup in a sampling tray within the automated analyzer,
transferring the urine to a cuvette mounted within the automated analyzer,
injecting at least one reagent composition in an aqueous medium into the
cuvette,
the reagent composition containing a buffer to adjust the pH of the urine
to a preferred value, a surfactant, at least two compounds to remove
substances in the urine that cause interference with colorimetric
photometry selected from the group consisting of 2,3-butanedione monoxime,
ethylenediaminetetraacetic acid, dimercaptopropanol, bile salts, albumin,
calcium chloride, peroxidase, hydrogen peroxide, dehydrogenase, 3-indolzol
acetate, N-toluene sulfonyl alanine indole ester and pyrrole amino acid
ester, together with a color indicator to quantitatively determine white
blood cells in the urine,
reading at specified intervals, in accordance with a preprogrammed code
introduced into the automated analyzer, at a preprogrammed
monochromatically specified wavelength, to compare absorbance of the
patient's urine and reagent composition complex with that of a standard
containing a known concentration of white blood cells and thereby
determining the presence or absence of white blood cells in the patient's
urine.
2. The method according to claim 1 wherein there is a first and second
reagent composition in an aqueous medium injected into the cuvette.
3. The method according to claim 1 wherein the wavelength of the analyzer
is about 405 nanometers.
4. The method according to claim 1 wherein said at least one reagent
composition further comprises a first reagent composition comprising a
buffer to adjust the pH of the urine to a preferred value, a surfactant,
2,3-butanedione and a compound to remove substances in the urine that
cause interference with colorimetric photometry further selected from the
group consisting of bile salts, albumin, calcium chloride,
dimercaptopropanol and ethylenediaminetetraacetic acid, and a second
reagent composition comprising a buffer, a surfactant, a color indicator
and a compound selected from the group consisting of dehydrogenase,
3-indolzol acetate, N-toluene sulfonyl alanine indole ester, and pyrrole
amino acid ester.
Description
BACKGROUND OF THE INVENTION
This invention relates to a method and materials that are designed for use
in automating urinalysis. This system is designed to analyze urine for its
constituents by a method that is fully automated (does not require the use
of manual methods such as refractometer, pH meter, dipsticks, etc).
Automation as designed by this system would be directed to the use of a
self-operating instrument that is capable of handling multiple reagents
designed for use on a automated analyzer system for the quantitative
determination of Leukocytes, Blood, pH, specific gravity, Glucose,
protein, Bacterial Nitrite, and Total Ketone Bodies in urine.
It is known that the most common method for the analysis of urine is by the
use of a manual technique known as a dipstick. This method for the
analysis of urine is labor, time intensive, and costly among other
detriments. The use of a dipstick for analysis of urine also relies on the
subjective interpretation of the technician. The dipstick method requires
the technician to submerge the dipstick in a sample of urine and remove
it. To wait a specified time, then compare the color development of the
test on the dipstick to a color chart. Even more cumbersome methods
involve the use of a refractometer, pH meter, or manual chemistry test.
The automated urinalysis system offers a method for reducing the
consumable materials, and labor costs. The system also offers increased
accuracy, sensitivity, and objective quantifiable determinations of
urinary constituents for better diagnostic interpretation of the test
results of urine, thus enabling a physician to provide better health care
for the patient.
In today's atmosphere of rising health care costs and the concomitant
reduction in the quality care, a system for the determination of normal or
disease state in urine that reduces the cost of testing by decreasing,
time, labor, and cost of test materials is called for. An obvious
advancement in the science of urinalysis would be to move it from the
slow, tedious, and costly techniques such as the dipstick or other manual
operations to a fully automateable system that would speed up
turn-around-time of obtaining test results, shorten doctor office visits,
and reduce overall health care costs. The automated urinalysis system
enables a technician to take a sample of urine and place it on an auto
analyzer that contains the system reagent materials for each of the above
referenced tests, start the self-operating analyzer, and walk away to
other duties until the results are reported by the analyzer. Most of the
high throughput analyzers currently in use throughout the industry would
allow testing in the aforementioned method of hundreds to thousands of
urines per hour. This is a marked improvement when compared to manual
techniques such as the dipstick which at best would take hours and several
technicians to analyze a hundred specimens. An article printed in the
American Journal of Clinical Pathology Vol. 83 pages 740-743, discusses
the cost of using the dipstick as a screening test for urinalysis. The
dipstick methodology can qualitatively determine normal urine
constituents. This article states "The urine dipstick procedure costs
approximately $0.76 for reagents and 3.0 minutes of the technologist's
time". This equates to 20 samples per hour with the use of dipsticks.
Another point to make here is that the cost has obviously risen since
1985. The time is now for the evolution of acceptable techniques for
determining normal and abnormal urinary constituents.
The following list of assay devices utilizing prior art includes dry
tablets, dipsticks, or other manual techniques for the analysis urinary
constituents. None of the prior devices foresee or teach of a
multiple/single liquid reagent system designed specifically for
auto-analyzers to analyze urinary constituents quantitatively.
One such U.S. Pat. No. 4,147,514 discloses test strips (dipsticks) for the
detection of ketone bodies. The assay strips are made up of a chemical
bonded to a cellulose pad on a strip. This is then dipped into a specimen
sample. This method only determines ketone bodies qualitatively at its
best, due to inability of the system to allow the use of standards and
controls on the same strip the sample is applied to. This assay does not
foresee or teach of a liquid reagent system that is designed to be pumped
by an autoanalyzer system into its discrete cuvette and there mixed with a
urine sample, and then measured by spectrophotometric means, and followed
by a computer driven calculation of a quantitative value derived from
standards previously run by the same analyzer. By utilizing an objective
instrument (autoanalyzer) which incorporates the use of standards and
controls instead of the subjective observation of the naked human eye with
no set reference point. The automated urinalysis system can elucidate
scientifically verifiable increases in accuracy, precision, and
sensitivity yielding quantitative reproducible results. The dipstick
method can not accommodate the use of standards, or controls with every
sampling of a urine for the calculation and verification of the result,
and thus limits its utility to producing a qualitative result only. Nor,
does the assay foresee or teach of the specific and unique chemical
formulation that the automated urinalysis reagent for ketone bodies is
comprised of. Obviously, many advances and differences exist between the
automated urinalysis system (herein described) and the prior art. This
automated system is a marked advancement in the art of urinalysis.
Another such patent, U.S. Pat. No. 3,146,070 discloses analytical
compositions in dry form on a bibulous carrier (dipstick) impregnated with
a pH indicator for the determination of pH. This assay at best only
determines pH qualitatively, due to the inability to use standards and
controls located on the same strip for the same test sample to define and
verify a quantitative determination. The assay does not foresee or teach
of a liquid reagent system that is designed to be pumped through an
auto-analyzer, mixed with urine in a discrete cuvette, measured by
spectrophotometric means, and automatically calculate a quantitative value
derived from standards run previously on the analyzer. This system also
allows for controls to be run periodically to insure continued precision
of results. The dipstick method can not accommodate the use of standards
or controls with every sampling of a urine for the calculation and
verification of result, and thus limits its utility to producing a
qualitative result with much less accuracy and precision than the
automated urinalysis system. As mentioned and to further illustrate, the
dipstick does not have the ability to sample a standard and the unknown
solution at the same time, and on the same dipstick, and thus allow
determination of a quantitative result. Nor does the assay foresee or
teach of the specific and unique chemical formulation that the automated
urinalysis reagent for pH is comprised of. Obviously there lies multiple
advances and differences that exist between the automated urinalysis
system and the prior art. This automated system exhibits a clear, obvious,
and marked advancement in the art of urinalysis.
Additionally U.S. Pat. No. 4,318,709 discloses a device comprising a
carrier matrix (dipstick) impregnated with the test means for specific
gravity. This assay at best only determines specific gravity
qualitatively, due to the inability to use standards and controls located
on the same strip for the same test specimen. The prior art in this case
also did not foresee the wide specimen to specimen matrix variations of
real world urine samples including matrix components such as pH, and ionic
strength, and the concomitant requirement of a multiple reagent system to
effectively analyze urine for specific gravity in a liquid to liquid
reaction. The normal pH value for urine can range from 4.5 to 8.0, which
if using the prior dipstick method the results would be vastly scattered
and inaccurate without a reagent to neutralize the effect prior to
completion of the assay. The assay does not foresee or teach of a liquid
reagent system that is designed to be pumped through an autoanalyzer,
mixed with urine in a discrete cuvette, and measured by spectrophotometric
means, and automatically calculate a quantitative value derived from
standards previously run on the analyzer. The dipstick method does not
have a means for the use of standards or controls with every sampling of a
urine for the calculation and verification of a result, and thus limits
its utility to producing a qualitative result with less accuracy and
precision than the automated urinalysis system. As mentioned and to
further illustrate, the dipstick does not have the ability to sample a
standard and unknown solution at the same time, and on the same dipstick,
and thus allow determination of a quantitative result. Nor, does the assay
foresee or teach of the specific and unique chemical formulation that the
automated urinalysis reagent for specific gravity is comprised of.
Obviously there lies multiple advances and differences that exist between
the automated urinalysis system and the prior art. This automated system
exhibits a clear, obvious, and marked advancement in the art of
urinalysis.
Various devices are described in the literature for the determination of
particular urinary constituents one by one with the use of carrier
matrices (dipstick, microcapusules, filter paper, etc.). None of the prior
art teaches or elucidates a means for determining by automated technology
urinary constituents from a single sample of urine, via multiple tests
that are reported simultaneously by an autoanalyser using liquid reagents
specifically designed for this family of instruments. As cited by the
prior art, (in package insert literature) when evaluating laboratory test
results, definitive diagnostic, or therapeutic decisions should not be
based on any single result or method. However, the prior art states that
dipsticks are affected by substances that cause abnormal urine color, such
as drugs containing azo dyes (e.g., Pyridium, Azo Gantrisin, Azo
Gantanol), nitrofurantoin (Macrodantin, Furadantin), and riboflavin, and
thus may affect the readability of reagent areas on the urinalysis reagent
strips (dipsticks). The color development on the reagent pad may be
masked, or a color reaction may be produced on the pad that could be
interpreted visually and/or instrumentally as a false positive or
negative. This illustrates the susceptibility of the prior art to
erroneous results due to misinterpretation of the color changes (due to
subjective observation by analyst), or interference with the reagent color
by urinary constituents that yield contradictory color changes, or
contamination from adjacent reaction pads spilling interfering color
and/or chemicals onto neighboring pads thus causing erroneous results
(from cross reaction, inhibition, or activation with test reagents
impregnated on test pads). Prior art does not envision or describe the
unique formulations needed for such analysis. Furthermore, the prior art
does not teach, describe, or elucidate, about a liquid reagent system
designed for liquid to liquid reactions without the use of a carrier
matrix. Finally, the prior art does not describe, teach, or elucidate, any
knowledge of this automated urinalysis system that is capable of the
analyzing unknown urine test samples at the same time as standards and
controls to allow for the extrapolation of accurate, and reproducible
quantitative values, yielding increases in accuracy, precision, and
sensitivity. Therefore, it is considered highly desirable to provide a
sensitive, rapid, accurate, reliable, time and cost saving, method and
device for the determination of urinary constituents. None of the prior
art known to the present inventors at the time of filing of the
application teaches or suggests the invention presently disclosed and
claimed.
OBJECTS AND SUMMARY OF THE INVENTION
In retrospect this invention is the answer to many of the problems
unanswered by the prior art: quantitative results, non-subjective results,
reproducible results, increased accuracy, precision, sensitivity, carrier
free reagents, reagents designed for autoanalyzer use, reagents uniquely
designed for each particular urine analyte assay overcoming matrix
problems previously unanswered by prior art, a method allowing vast
improvement of test completion time (hundreds to thousands per hour). The
present invention presents a fully automateable walk-away urinalysis
system applicable to any discrete autoanalyzer currently in use, and
obviously represents a marked advancement in art of urinalysis. The clear
cut object of the present invention is to provide a more comprehensive
method for determining urinary constituents (Leukocytes, Blood, Bacterial
Nitrite/Indole/reductase activity, Total Ketone Bodies, Glucose, Protein,
pH, and specific gravity) that in general benefit society as a whole and
specifically yield improved health care.
Thus, it is a primary objective of the present invention to provide
techniques for determination of urinary constituents (Blood (Red Blood
Cells/Hemoglobin), Leukocytes, pH, Specific Gravity, Bacterial
Reductase/Indole/Nitrite activity, Total Ketone Bodies, Protein, and
Glucose) at low chemically significant levels. These methods must be fast,
efficient, adaptable to many of the currently available discrete and
continuous flow automated analyzers, effective at sample to reagent ratios
of 1 to 13 or more (unlike like the prior art, because this ability is
essential for application to most autoanalyzers), and applicable to
samples with high turbidity, high ionic strength, high color content, and
wide pH extremes, and buffer strengths among other interfering substances.
An additional object of this invention is to make available an advanced
method for analyzing a sample of urine for the quantitation of its
constituents on an autoanalyzer. The advanced ability of the automated
urinalysis system to offer a means for automated analysis on urine is a
significant improvement the in art of urinalysis.
Additionally, the object of this invention is to provide a comprehensive
method which is broadly adaptable to a wide variety of automated analyzers
presently in use in the industry which will increase accuracy,
sensitivity, precision, and speed. An autoanalyzer would also allow for
precise quantitative results which are beyond the scope and abilities of
the prior art. An autoanalyzer used in conjunction with the present
invention automated urinalysis reagents would also provide a system that
can produce an objective quantitative result of an unknown urine sample
obtained from a linear standard curve determined by analysis of standards
run on the instrument, and verified as accurate by quantifying controls of
known value. This simultaneous analysis of standards and unknowns (urine
samples) yielding unbiased results would improve the art of urinalysis
significantly over the prior art, which yields only qualitative and
subjective results.
It is a further object of this invention to provide a method for the
simultaneous determination of multiple urinary components (Leukocytes,
Blood, Bacterial Reductase/Nitrite/Indole activity, Total Ketone Bodies,
pH, Specific Gravity, Protein) from a single urine sample using a system
of reagents designed for autoanalyzer use. This improvement in the science
of urinalysis over the prior art will prove to be significant medically
and economically.
Another object of this invention is to provide a method that yields
quantifiable results in the determination of urinary constituents present
in a sample of urine. None of the prior art teaches, elucidates, or
envisions a method for the determination of quantitative values for
urinary constituents: Leukocytes, Blood, Bacterial
reductase,/Nitrite/Indole activity, Total Ketone Bodies, pH, specific
gravity, Glucose, Protein. The prior art can provide only qualitative
results. For example, using current art a technician must dip a urine
stick into a sample, remove, observe and record color changes for eight
separate test blocks on the strip. Each of these eight tests require
accurate, precise, and specific and different times for color development,
and the technologist must measure them accurately while judging and
recording the relative intensities of various shades of color. This
obviously cumbersome, time intensive, subjective, inaccurate, method can
vastly be improved upon by the use of the present invention.
Still another object of this invention is to provide a method for the
determination of objective results (from the photometric analysis by the
automated analyzer) instead of the subjective determination (from human
observation). The present invention provides a unique formulated reagent
system that can be mixed with unknown urine samples, standards, and
controls and then be read spectrophotometrically with unbiased accuracy on
an autoanalyzer. The use of the automated urinalysis system provides a
means for improved accuracy, precision, and specificity by removal for the
subjective human element from the analysis. Clearly, a system that
automatically dispenses, measures, and records results is a marked
improvement in the science of urinalysis.
Yet another object of this invention is to provide uniquely formulated
reagents for each urinalysis assay that were not taught or envisioned by
the prior art, and overcome the inadequacies of the prior art. The
analysis for Blood in urine in the prior art is a carrier dependent assay
that is susceptible to interference urea, vitamin C, and high levels of
some other normal urinary constituents. Consider the fact that urea is the
largest component of urine (besides water) by a factor of 50% over the
next largest component (sodium chloride). A unique chemical formulation to
compensate for urea would be an advancement in the art of urinalysis. The
present invention is a liquid reagent that is not carrier dependent,
designed for autoanalysis, and has agents added to remove the urea and
other interfering ions from the solution, thus preventing it from
interacting with the color developer. These improvements increase
sensitivity, accuracy, and precision, thereby allowing the Blood assay in
urine to be quantifiable.
Yet another object of this invention is to provide uniquely formulated
reagents for each automated urinalysis assay that was not taught or
envisioned by the prior art, and overcomes the inadequacies of the prior
art. The assay for pH in the prior art is limited to a carrier dependent
assay (i.e., solid matrix), and its sensitivity is limited to qualitative
whole number units. It has a non-specific s-shaped curve with 7 color
changes for determination of pH within the very small range of pH 5 to pH
8.0. These different color changes make analysis by an autoanalyzer's
single wavelength (monochromatic) spectrometry impossible. Another problem
with this assay is the inaccuracy introduced by the subjective
interpretation of changes in color gradations and shades by the technician
and the inability of color-blind people to perform the test. The prior art
is a matrix dependent method that cannot be used in a carrier free liquid
reagent system designed for autoanalysis. The multitude of color changes
including orange, yellow, blue, green, and intermediates shades make the
use of the prior art impossible for quantitative, sensitive, accurate, and
precise monochromatic spectrophotometric analysis. The present invention
is a liquid reagent that is not carrier dependent, and is designed for use
on autoanalyzers. The present invention is linear from pH of 3.0 to a pH
of greater than 10.0. The present invention has a curve stabilizer added
to increase curve stability and to provide a flat line analysis, thus
removing the s-shaped curve phenomena. The present invention is
quantifiable to within 0.01 pH units. It is more precise, accurate, and
sensitive than the prior art, and thus represents an obvious advancement
in the art of urinalysis.
Yet another object of this invention is to provide uniquely formulated
reagents for each automated urinalysis assay that was not taught or
envisioned by the prior art. The assay for Leukocytes in the prior art has
limited accuracy and application because it is carrier dependent, it only
produces qualitative results (i.e., trace, 1+, 2++, 3+++, or a range 5 to
15 leukocytes present), it yields numerous color changes (5) making
objective monochromatic spectrophotometric analysis impossible, and it
cannot be easily and effectively converted to a liquid matrix, which is
required for widespread autoanalyzer use. The prior art is susceptible to
interference from sample urine matrices including but not limited to high
ionic strength, antibiotics, and glucose. The prior also takes a minimum
of 2 minutes for color development and subjective interpretation of
results. The present invention is a liquid reagent that is not carrier
dependent, and is specifically designed for use on autoanalyzers. The
present invention is quantitatively linear from 0.0 esterase units of
activity to greater than a 100 esterase units of activity (0 to 25
Leukocytes and greater). The present invention directly measures the
amount of leukocytes present by quantitatively measuring the leukocyte
esterase activity in urine. This is accomplished by a colormetric reagent
specifically designed for use on an autoanalyzer, and is sensitive to
leukocyte esterase. The present invention includes a compensator (buffer)
for adjusting the pH of the urine samples because random samplings can
range from 4.5 to 8.0. Buffering the sample is critical to obtaining
optimal sensitivity, and precision because Leukocyte esterase activity is
optimal at a pH of 6.8. Due to its solid matrix the prior art is incapable
of compensating for abnormal pH resulting in its poor sensitivity and
precision. The present invention has curve stabilizers and agents added to
compensate for the wide variety of interfering substances found in urine,
which the prior art does not teach or envision. The present invention is
quantitative, carrier independent, precise, accurate, and sensitive, and
would be an advancement in the art of urinalysis.
Yet another object of this invention is to provide uniquely formulated
reagents for each automated urinalysis assay that was not taught or
envisioned by the prior art. The assay for Bacterial
reductase/Nitrite/Indole activity, in the prior art has limited
application and accuracy because it is carrier dependent, and it only
produces qualitative results (i.e., positive or negative with a range of
0.06 to 0.1 mg/dl of nitrite ions present). The measurement of nitrite is
a indirect method suggesting the presence of gram negative micro organisms
that reduce nitrate to nitrite. Urinary tract infections can occur from
organisms that do not convert nitrate to nitrite (i.e., gram positive),
thus a false negative would occur. If dietary nitrate were absent, the
gram negative bacteria could not make nitrite again resulting in a false
negative test. If the urine is not held in the bladder for at least 4
hours a false negative can again result, because the bacteria require this
time to convert nitrate to nitrite in sufficient quantities for detection.
It should be noted that frequent urination is often associated with
bacterial urinary infection. The prior method yields a non-specific color
development for determination of Nitrite present making objective and
monochromatic spectrophotometric analysis difficult. Extrapolation of
prior to the present invention is not readily apparent to anyone schooled
in the art of urinalysis. The prior art is susceptible to interferences
from sample matrices including, but not limited to high ionic strength and
Vitamin C. The present invention is a liquid reagent that is not carrier
dependent, and is specifically designed for use on autoanalyzers. The
present invention is quantitatively linear from 0.05 mg/dl to 1.0 mg/dl
nitrite ions present. The present invention also directly measures
quantitatively the amount of reductase present (which is the enzyme
present that converts nitrate to nitrite). There are several advantages to
measuring the reductase including, but not limited to more direct
measurement of bacteria present, bladder incubation time not required, and
resulting assay is more accurate, sensitive, and quantitative. The present
invention utilizes colormetric reagents specifically designed for
autoanalyzer, and can directly measure the amount of nitrite ion, indole
activity, or reductase present. The present invention has a compensator
for the pH of the random urine sample which can range from 4.5 to 8.0. It
should be noted that Nitrate reductase activity is optimal at a pH of 6.8.
Buffering the sample to this pH is critical to obtaining optimal
sensitivity, accuracy, and precision. The present invention measures the
activity of nitrate reductase on nitrate (substrate) by the disappearance
of NADPH which absorbance can be monitored at 340 nm. The prior art has no
means to compensate for abnormal pH, resulting in poor sensitivity and
selectivity of the assay. The present invention has curve stabilizers and
agents to compensate for a variety of interfering substances found in
urine, which the prior art did not teach or envision. The present
invention is quantitative, carrier independent, precise, accurate,
automateable, and sensitive, and represents an obvious advancement in the
art of urinalysis.
Another object of this invention is to provide uniquely formulated reagents
for each automated urinalysis assay that was not taught or envisioned by
the prior art. The assay for specific gravity in the prior art has limited
accuracy and application because it is a carrier dependent assay, and it
only produces semi-qualitative results ranging from 1.000 to 1.030
specific gravity units in increments of 5 specific gravity units (i.e.,
1.000, 1.00, 1.010, 1.015, 1.020 . . . ). The prior art can not
extrapolate a more sensitive quantitative value (i.e., 1.003, 1.004, . . .
). The prior method produces a multitude of changes in color gradations
and shades (at least 7 different color changes) making accurate, precise,
objective, monochromatic spectrophotometric autoanalysis impossible.
Someone skilled in the prior art could not convert it to a liquid matrix
as required for use on autoanalyzers. The prior art is susceptible to
interferences from sample matrices including, but not limited to high or
low pH, elevated urinary protein, and highly buffered urines. The prior
art also requires 45 seconds incubation period for test completion
increasing the chance of operator error and cost of testing. The present
invention is a liquid reagent that is not carrier dependent, and is
specifically designed for use on autoanalyzers. The present invention is
quantitatively linear from 1.000 to 1.050 with precision of plus or minus
0.0005 specific gravity units. The present invention is a colormetric
reagent system specifically designed for autoanalyzer use that is
sensitive to ions in solution. The present invention has a compensator for
highly buffered urines, and diverse urinary pH which can range from 4.5 to
8.0 in random urines. The prior art did not teach or elucidate a method to
neutralize the pH and buffer activity of a urine prior to assaying for ion
content. This failure of the prior art to compensate for abnormal pH
directly contributes to its poor accuracy and precision. The present
invention has curve stabilizers and agents added to compensate for the
wide variety of interfering substances found in urine, which the prior art
did not teach or envision. The present invention is quantitative, carrier
independent, precise, accurate, and sensitive, and is an obvious
advancement in the art of urinalysis.
Again, another object of this invention is to provide uniquely formulated
reagents for each automated urinalysis assay that was not taught or
envisioned by the prior art. The assay for Total Ketone Bodies in the
prior art has limited accuracy and application because it is a carrier
dependent assay and it only produces semi-qualitative results ranging from
5 to 10 mg/dl acetoacetic acid. The prior method produces a multitude of
changes in color gradations and shades (at least 6 different colors) for
determination of ketone bodies making accurate, precise, and monochromatic
spectrophotometric autoanalysis impossible. Some one skilled in the prior
art could not easily and effectively convert it to a liquid matrix, as
required for use on an autoanalyzer. The prior art is qualitative and only
measures acetoacetic acid which constitutes only 20% of the total ketone
bodies present in urine. Please note that B-Hydroxybutyric acid makes up
approximately 80% of the ketone bodies present in urine. The prior art is
susceptible to interferences from sample matrices including, but not
limited to highly pigmented urines, sulfhydryl groups (causing false
positive results), high or low pH values, levodopa metabolites, mesna
(2-mercaptoethane sulfonic acid) causing false positive results, atypical
color development and high ionic strength urines. The prior art also
requires 40 seconds incubation period for test completion increasing the
chance of operator error, and cost of testing. The present invention is a
liquid reagent that is not carrier dependent, and is specifically designed
for use on autoanalyzers. The present invention is quantitatively linear
from 0.0 to 25 mg/dl of acetoacetic acid or greater in increments of 0.1
mg/dl. The present invention also measures quantitatively the amount of
B-hydroxybutyric acid present with a sensitivity range of 0.0 mg/dl to 100
mg/dl B-hydroxybutyric acid. This is done by the use of a colormetric
reagents specifically designed for autoanalyzer use that are sensitive to
the presence of acetoacetic acid and B-Hydroxybutyric acid in solution.
The present invention has a compensator for highly buffered urines, and
diverse urinary pH which can range from a pH of 4.5 to 8.0 in random
urines. The prior art did not teach of or elucidate a method to neutralize
the pH and ionic content of a urine prior to assaying acetoacetic acid
content. This failure of the prior art to compensate for abnormal pH and
buffering directly contributes to its poor accuracy and precision. This
lack of precision and accuracy of the prior art is also directly
attributable to its lack of sensitivity to B-Hydroxybutyric acid, the
major component of ketone bodies present in urine. The present invention
has curve stabilizers and agents added to compensate for the wide variety
of interfering substances found in urine, which the prior art did not
teach or envision. The present invention measures the presence of
B-hydroxybutyric acid in urine at the same time or separately with
acetoacetic acid quantitation, thus greatly enhancing its accuracy and
precision. The present invention is quantitative, carrier independent,
precise, accurate, and sensitive, and represents an obvious advancement in
the art of urinalysis.
Yet another object of this invention is to provide uniquely formulated
reagents for each automated urinalysis assay that were not taught or
envisioned by the prior art. The assay for Protein in the prior art is
limited to a carrier dependent assay that is only semi-qualitative
producing results ranging from 15 to 30 mg/dl protein. The prior method
yields non-specific color development with more than 6 different colors
changes for determination of protein making objective and monochromatic
spectrophotometric analysis impossible. Someone skilled in the art cannot
easily elucidate or convert the prior art into the matrix required for use
on autoanalyzers. The prior art only semi-qualitatively measures protein
in the form of albumin which constitutes only 30% of the urinary protein
excreted in urine. Please note that the majority of protein excreted in
urine is in the form of globulins. The prior art is also susceptible to
interference due to, but not limited to highly buffered urine, urine with
high pH values, quaternary ammonium compounds (i.e., from some antiseptics
and detergents) or skin cleaners containing chlorhexidine, and other
normal urinary constituents. The reagent and high ionic strength urine.
The prior art also requires a carefully measured 60 second period to
obtain correct analytical results. The present invention is a liquid
reagent that is not carrier dependent, and is specifically designed for
use on most currently available autoanalyzers. The present invention is
quantitatively linear from 0.0 to 100 mg/dl of protein with precision of
0.1 mg/dl. The present invention quantitatively measures the amount of
globulin accounting for approximately 70% in urine, and albumin
(approximately 30%) accurately in the range of 0.0 mg/dl to 100 mg/dl.
This is done by the use of a colormetric reagents specifically designed
for autoanalyzer use and are sensitive to protein in the form of albumin
and globulins. The present invention has a compensator for pH (which can
range from a pH of 4.5 to 8.0), and highly buffered urines. The prior art
did not teach of or elucidate a method that would neutralize the pH and
ionic strength of a urine prior to analysis of protein content. The prior
art has no means to compensate for abnormal pH, contributing to its poor
sensitivity and selectivity to the presence of protein in the urinary
sample matrix. The present invention has curve stabilizers and agents
added to compensate for the wide variety of interfering substances found
in urine. These innovations were not taught or envisioned by the prior
art. The present invention measures the quantity of albumin and globulin
in urine simultaneously, or separately. The present invention is
quantifiable, carrier independent, precise, accurate, and sensitive
method, and represents an obvious advancement in the art of urinalysis.
Yet another object of this invention is to provide uniquely formulated
reagents for each automated urinalysis assay that was not taught or
envisioned by the prior art. The assay for Glucose in the prior art is
limited to a carrier dependent assay that produces only qualitative
results ranging from 75 to 125 mg/dl of glucose. The prior method yields
color development with more than 6 different color changes for the
determination of glucose making subjective, monochromatic, and
spectrophotometric analysis impossible. Furthermore, someone skilled in
the art cannot easily elucidate or converts the prior art into the liquid
matrix required for use on autoanalyzers. The prior art only qualitatively
measures glucose. The prior art is also susceptible to interference from,
but is not limited to high ionic strength urines, Vitamin C, and Ketone
Bodies. The prior art also requires a carefully measured 30 second
incubation period to obtain correct semi-qualitative results. The present
invention is a liquid reagent that is not carrier dependent, and is
specifically designed for use on autoanalyzers. The present invention is
quantitatively linear from 0.0 to 250 mg/dl glucose with precision to 0.1
mg/dl. The present invention also quantitatively measures the amount of
glucose. This is done by the use of a colormetric reagents that are
sensitive to the presence of urinary glucose, and are specifically
designed for use on autoanalyzers. The present invention has a compensator
for the pH (which can range from a pH of 4.5 to 8.0 in random urines). The
prior art did not teach, or elucidate a method that would neutralize the
pH and ionic strength of a urine prior to analysis of glucose content. The
prior art has no means to compensate for abnormal specific gravity, which
contributes to its poor sensitivity and selectivity to the presence of
glucose in the urinary sample matrix. The present invention has curve
stabilizers and agents added to compensate for the wide variety of
interfering substances found in urine, which the prior art did not teach
or envision. The present invention is quantitative, carrier independent,
precise, accurate, and sensitive, and represents an obvious advancement in
the art of urinalysis.
Other objects and a fuller understanding of the invention will be had by
referring to the following description and claims of the preferred
embodiment and will become apparent to those skilled in the art.
DETAILED DESCRIPTION OF THE INVENTION
The presently claimed method comprises a group of carrier-free liquid
reagents designed for simultaneous usage on automated analyzers for
quantitative determination of urinary constituents. The automated
urinalysis system of the instant invention solves the problems confronting
automating the analysis of urine, and in the process represents a
significant improvement over the present art. These improvements which
permit (facilitate) application to automation and represent significant
technical improvement over the previous art include, a buffering system
for pH variation in urine by correcting pH to the analytically preferred
value prior to analysis, and also stabilizing reaction rates thereby
improving linearity and neutralizing the interference effects of the
highly complex matrix of random urines submitted for analysis. Additional
technical improvement is due to the addition of components to remove
interfering substances yielding reduced assay limitations and increased
linearity, accuracy, and precision in the resulting quantitations. These
unique reagent formulations allow automation resulting in (but not limited
to) enhanced, speed, objectivity, accuracy, and sensitivity associated
with a synopsis of the automated testing process follows. The entire
automated urinalysis reagent system is then loaded into an autoanalyzer,
the controls, standards, and unknown urine samples are fed into the
autoanalyzer, individually mixed with each test reagent in discrete
cuvettes, the absorbance read, and quantitation determined for comparison
with the standard curve.
The composition of each reagent of the present invention is designed for
optimum reaction with the random urine samples and to effectively deal
with problems arising from the tremendous variability from sample to
sample due to the diet, disease state, medications, time of collection,
state of hydration, sex, age, and physical well being of the patient. All
of the factors can interfere with the previous art.
The automated urinalysis system reagents are individually designed for
optimum analysis of the specific urinary component. The reagent system to
detect Blood (RBC's)in urine is carrier-independent, and contains specific
agents added to compensate for interference by urea, vitamin C, high ionic
levels (specific gravity), abnormal pH, and other normal urinary
constituents. The RBC reagent system is composed of two reagents (but can
be consolidated into one). The first reagent (R1) is specifically designed
to neutralize matrix interference and increase sample-reagent
compatibility, with the autoanalyzer. 2,3-Butanedione monoxime is added to
the first reagent (R1) to remove urea, and other substances in the urine
sample that cause interference with colormetric reactions utilizing any of
the following components 3,3',5,5'-Tetramethylbenzidine, Dicarboxidine, 3-
Methyl-2- benzothiazolinone hydrazone, or N,N- dimethylaniline. The
components listed above are particularly susceptible to interference from
urea (a major component of urine). Ethylenediaminetetraacetic acid
(disodium salt) and dimercaptopropanol are other components of the R1 used
to neutralize interfering substances by chelation, and anti-oxidant
activity. This compound removes oxidizing contaminants such as
hypochlorite, and acts as a solution clarifyer (it causes the
disappearance of the characteristic yellow color of urine), thereby
enhancing spectrophotometric analysis. 2,3-Diphosphoglycerate is added to
affect the oxygen dissociation of hemoglobin. Saponin is present to lyse
the red blood cells that may be present and intact in urine, thus
releasing the hemoglobin contained within. Note that
2,3-Diphosphoglycerate in the alkaline reagent mixture causes the
dissociation constant of hemoglobin to shift to the left (acid Bohr
effect), thus increasing the affinity of hemoglobin for oxygen and forcing
the reaction to completion. Oxygen is provided by the reaction of
hemoglobin with hydrogen peroxide. Sodium azide is added to stabilize
hydrogen peroxide. The R1 contains hydrogen peroxide acting as a substrate
for the peroxidase activity of the heme fraction of hemoglobin which is a
major component of red blood cells. The R1 also contains a buffer to
adjust sample pH and aid in solubility and compatibility R1's complex
chemical matrix. This complex reagent matrix requires a complementary
buffering system with unique dynamics, capable of adjusting the reaction
solution to the ideal pKa, and promoting component solution compatibility
in an aqueous medium autoanalyzers. Unbuffered solutions may have high
acidic or basic activity, or strictly organic properties which are not
compatible with autoanalyzer syringes, tubing, metal, and plastic parts.
This reagent system buffer is designed to correct these problems. The
buffers also promote carrier independence. The R1 also contains
surfactants that decrease surface tension, promote effective mixing on a
molecular level, and improve flow dynamics through tubing and syringes of
automated analyzers. The concentrations of R1 buffers and components can
be varied to compensate for limitations and variations in the
configuration of sampling and reagent delivery systems of various makes of
autoanalyzers. The R1 components compensate for abnormal urinary pH, and
highly buffered urines. Ampyrone is added to the R1 to promote, or
catalyze the reaction of the afore mentioned oxidized peroxide molecule
with a coupling agent such as p-hydroxybenzoic,
N-Ethyl-N-sulfohydroxypropyl-m-toluidine (TOOS),
2-Hydroxy-3,5-dichlorobenzenesulfonate sodium salt (HDCBS),
2,2'-Azino-di-3-ethylbenzthiazoline sulfonic acid diammonium salt (ABTS),
or trinder, or phenolic substitutes. The addition of Pyrogallol is added
to R1 and acts as a substrate that is oxidized by the oxygen radical
released when the heme (peroxidase active) molecule reacts with hydrogen
peroxide in solution.
The second reagent (R2) of the 2 part reagent system for Blood (if a single
reagent system for Blood is not used) is composed of one, or more of the
following: 3,3',5,5'-tetramethylbenzidine, dicarboxidine, pyrogallol,
hydrogen peroxide, 3-methyl-2-benzothiazone hydrazone,
N,N-dimethylaniline, benzidine, o-dianisidine, and oxidized phenothiazines
in solution. This reagent is buffered according to which group or single
component is used. This buffer contained in R2 adjusts sample pH and aids
in solubility and compatibility of R2's complex chemical matrix. This
complex reagent matrix requires a complementary buffering system with
unique dynamics capable of adjusting the reaction solution to the ideal
pKa's, establishing carrier independence, and promoting component solution
compatibility in an aqueous medium with autoanalyzers. Unbuffered
solutions may have high acidic or basic activity, or strictly organic
solubilities properties which are not compatible with autoanalyzer
syringes, tubing, metal, and plastic parts. The R2 also contains
surfactants that decrease surface tension, promote effective mixing on a
molecular level, enhance carrier independence, and improve flow dynamics
through tubing and syringes of automated analyzers. The combinations and
concentrations of R1 and or the R2 components can be varied due to
limitations and variations in the configuration of sampling and reagent
delivery systems of different makes of autoanalyzers. Without further
elaboration, it is believed that one skilled in the art can, using the
preceding description, effectively utilize the present invention. The
following preferred specific embodiments are, therefore, to be merely
illustrative, and not limitive of the remainder of the disclosure of the
present invention in any way whatsoever. In the following examples, all
instrument parameters, reagent combinations, and method techniques are
generalized.
EXAMPLE 1
The automated RBC urinalysis reagent system's first reagent (R1) contains
surfactant, 2,3-Butanedione monoxime, ethylenediametetraacetic acid,
dimercaptopropanol, saponin, 2,3- Diphosphoglycerate, and buffer. The
second reagent (R2) consists of surfactant, buffer,
3,3',5,5'-tetramethylbenzidine in 10% lactic acid. These reagents are
placed in the autoanalyzer. The urine samples, standards, and controls are
placed in the autoanalyzer specimen cups. The urine samples, standards,
and controls, are aliquoted into cuvettes, mixed with the first
reagent,and than mixed with the second reagent, and then read at specified
intervals as dictated by the instrument parameters, and at the specified
wavelengths (monochromatically) depending on reagent combination used. In
this instance the assay should be read at 660 nanometers with read times
specific to the analyzer.
EXAMPLE 2
The automated RBC urinalysis single reagent system would contain (all or
some of the following:) 2,3-Butanedione monoxime, ethylenediametetraacetic
acid, dimercaptopropanol, 2,3-Diphosphoglycerate, Ampyrone, Sodium azide,
hydrogen, peroxide, saponin, p-Hydroxybenzoic acid,
N-Ethyl-N-sulfohydroxypropyl-m-toluidine, surfactants, The reagents are
placed on the autoanalyzer. The urine samples, standards, and controls are
placed in the autoanalyzer specimen cups. The urine samples, standards,
and controls, are aliquoted into cuvettes, mixed with the reagent, and the
solutions are read at specified intervals as dictated by the instrument
parameters and the specified wavelength (monochromatically) depending on
the reagent combination used. In this instance, the assay should be read
at 505 nanometers read times are specific to the analyzer.
EXAMPLE 3
In the automated RBC urinalysis reagent system, first reagent (R1),
contains surfactants, buffer, 2,3- Butanedione monoxime,
dimercaptopropanol, saponin, 2,3- Diphosphoglucerate, and
ethylenediametetraacetic acid. The second reagent (R2) consists of,
hydrogen peroxide, Sodium azide, 3-methyl-2-benzothiazoline hydrazone,
N,N-dimethylanilane, buffers, and surfactants. The reagents are placed on
the autoanalyzer. The urine samples, standards, and controls are placed in
the autoanalyzer specimen cups. The urine samples, standards, and
controls, are aliquoted into cuvettes, with the first reagent, the second
reagent is then added and mixed, and the solutions are then read at
specified intervals as dictated by the instrument parameters at the
specified wavelength (monochromatically) depending on the reagent
combination used. In this instance the assay should be read at 585
nanometers and read times are specific to the analyzer.
EXAMPLE 4
In the automated RBC urinalysis reagent system's first reagent (R1)
contains surfactant, 2,3-Butanedione monoxime, ethylenediametetraacetic
acid, dimercaptopropanol, saponin, 2,3-Diphosphoglycerate, and buffer. The
second reagent R2 consist of surfactant, buffer, o-dianisidine. The
reagents are placed on the autoanalyzer. The urine sample, standards, and
controls are placed in the autoanalyzer specimen cups. The urine samples,
standards, and controls are aliquoted into cuvettes, mixed with the first
reagent, the second reagent is then added and mixed, and the solutions are
then read at specified intervals as dictated by the instrument parameters
at the specified wavelength (monochromatically) depending on the reagent
combination used. In this instance the assay should be read at 540
nanometers and read times are specific to the analyzer.
EXAMPLE 5
In the automated RBC urinalysis single reagent system would contain (all or
some of the following:), 2,3-Butanedione monoxime, Pyrogallol,
ethylenediametetraacetic acid, dimercaptopropanol, p-hydroxybenzoic acid,
saponin, 2,3- Diphosphoglycerate, Sodium azide, hydrogen peroxide,
N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine, surfactants, are added.
The reagents are placed on the autoanalyzer. The urine samples, standards,
and controls are placed in the autoanalyzer specimen cups. The urine
samples, standards, and control, are aliquoted into cuvettes, mixed with
the reagent, and the solutions are read at specified intervals as dictated
by the instrument parameters at the specified wavelength
(monochromatically) depending on reagent combination used. In this
instance, the assay should be read at 550 and read time is specific to the
analyzer.
EXAMPLE 6
In the automated RBC urinalysis reagent system's first reagent (R1)
contains surfactant, 2,3-Butanedione monoxime, ethylenediametetraacetic
acid, dimercaptopropanol, saponin, 2,3-Diphosphoglycerate, and buffer. The
second reagent R2 consist of surfactant, buffer, oxidized phenothiazines.
The reagents are placed on the autoanalyzer. The urine samples, standards,
and controls are placed in the autoanalyzer specimen cups. The urine
samples, standards, and controls are aliquoted into cuvettes, mixed with
the first reagent, then the second reagent is then added and mixed, and
the solutions are then read at specified intervals as dictated by the
instrument parameters at the specified wavelength (monochromatically)
depending on the reagent combination used. In this instance the assay
should be read at 540 nanometers and read times are specific to the
analyzer.
EXAMPLE 7
In the automated RBC urinalysis reagent system's first reagent (R1)
contains surfactant, 2,3-Butanedione monoxime, ethylenediametetraacetic
acid, dimercaptopropanol, saponin, 2,3-Diphosphoglycerate, hydrogen
peroxide, sodium azide, and buffer. The second reagent R2 consist of
surfactant, buffer, Ampyrone, p-Hydroxybenzoic acid, and phenol. The
reagents are placed on the autoanalyzer. The urine samples, standards, and
controls are placed in the autoanalyzer specimen cups. The urine samples,
standards, and control are aliquoted into cuvettes, mixed with the first
reagent, the second reagent is then added and mixed, and the solutions are
then read at specified intervals as dictated by the instrument parameters
at the specified wavelength (monochromatically) depending on the reagent
combination used. In this instance the assay should be read at 505
nanometers and read times are specific to the analyzer.
EXAMPLE 8
In the automated RBC urinalysis reagent system's first reagent (R1)
contains 2,3-Butanedione monoxime, ethylenediametetraacetic acid,
dimercaptopropanol, 2,3-Diphosphoglycerate, Sodium azide, hydrogen
peroxide, saponin, surfactants,and buffers. The second reagent (R2) has
buffers, surfactants, N-Ethyl-N-sulfohydroxypropyl-m-toluidine. The
reagents are placed on the autoanalyzer. The urine samples, standards, and
controls are placed in the autoanalyzer specimen cups. The urine samples,
standards, and control, are aliquoted into cuvettes, mixed with the first
reagent, the second reagent is then added and mixed, and the solutions are
then read at specified intervals as dictated by the instrument parameters
at the specified wavelength (monochromatically) depending on the reagent
combination used. In this instance the assay should be read at 550
nanometers and read times are specific to the analyzer.
EXAMPLE 9
In the automated RBC urinalysis reagent system's first reagent (R1)
contains 2,3-Butanedione monoxime, ethylenediametetraacetic acid,
dimercaptopropanol, 2,3-Diphosphoglycerate, Sodium azide, hydrogen
peroxide, saponin, surfactants, buffers. The second reagent (R2) consists
of buffers, surfactants, N-Ethyl-N-sulfohydroxypropyl-m-toluidine (TOOS),
and/or (one or more from the following group:
2,2'Azino-di-(3-ethylbenzthiazoline sulfonic diammonium salt (ABTS),
2-Hydroxy-3,5-dichlorobenzenesulfonate sodium salt (HDCBS), or other
suitable trinder reagent). The reagents are placed on the autoanalyzer.
The urine samples, standards, and controls are placed in the autoanalyzer
specimen cups. The urine samples, standards, and controls are aliquoted
into cuvettes mixed with the first reagent, the second reagent is then
added and mixed, and the solutions are then read at specified intervals as
dictated by the instrument parameters at the specified wavelength
(monochromatically) depending on the reagent combination used. In this
instance the assay should be read at 550 nanometers secondary wavelength
and read times are specific to the analyzer.
The automated urinalysis system reagents are individually designed for
optimum analysis of specific urinary components. The reagent system for
Leukocytes (WBC) in urine is carrier-independent, and has specific agents
added to compensate for interference caused by enzyme inhibitors, oxalic
acid, high ionic strength urines (specific gravity), glucose, antibiotics
(Tetracycline), cephalexin, cephalothin, abnormal pH values, and other
normal urinary constituents. The reagent system is composed of a single
reagent (but can be a two reagent system). This reagent system is
specifically designed for matrix interference neutralization, and
automated liquid reagent compatibility. The component 2,3-Butanedione
monoxime is included in this reagent to remove urea, and other substances
found in urine that cause interference with the colormetric reactions.
Examples: of interference include free radical oxidation of 3-indoyl
acetate, p-nitrophenyl stearate, phenyl laurate, N-toluene sulfonyl
alanine indole ester, derivatized pyrrole amino acid ester, or other
active esters in random urine specimens due to many components found in
Ethylenediaminetetraacetic acid (disodium salt) and dimercaptopropanol are
components added to the reagent and used to neutralize interfering
substances by chelating, remove enzyme inhibitors, and anti-oxidant
activity. This removes oxidizing contaminants such as hypochlorite and
heavy metals which are enzyme inhibitors, and act as a solution clarifyer
(it causes the disappearance of the characteristic yellow color of urine)
thereby enhancing spectrophotometric analysis. These interference
neutralizing compounds can be added to the reagent to react competitively
with the interfering substances, and enhance Leukocyte esterase activity.
The reagent may also contain bile salts, albumin and calcium ions (calcium
chloride) to increase esterase activity. Other enzyme activators are added
such as Calcium chloride (or other as magnesium chloride etc.). These
agents act to inhance activity of the esterase as well as prevent
denaturation of the enzyme. The reagent may also contain hydrogen peroxide
as a substrate (oxygen donor) for peroxidase. Peroxidase and hydrogen
peroxide intreact to yeild an oxygen radical. This radical acts to enhance
the color developing properties (speed, completeness, of reaction, ect.)
of the reagent system. Sodium azide is present as a hydrogen peroxide
stabilizer. The reagent also contains a buffer to adjust sample pH and aid
in solubility, and compatibility of the reagents complex chemical matrix.
This complex chemical matrix requires a complementary, aqueous buffering
system with unique dynamics capable of adjusting the reaction solution to
the ideal pKa, and promoting reagent component compatibility with
autoanalyzers. Unbuffered solutions may have high acidic or basic
activity, or strictly organic properties which are not compatible with
autoanalyzer syringes, tubing, metal, and plastic parts. This reagent also
contains surfactants that enhances the carrier-free matrix, decrease
surface tension, promote effective mixing on a molecular level, and
improve flow dynamics through tubing and syringes of automated analyzers.
The concentration of reagent buffers, and other components, can be varied
to compensate for limitations and variations in the configuration of
sampling and reagent delivery systems of various makes of autoanalyzers.
The reagent buffers also compensate for abnormal pH of urine samples and
urines with high buffer capacities.
The reagent system for Leukocytes (WBC's) may consist of a single reagent,
or a dual reagent system. The color generating mechanism of the reagent
system is the same for the single or dual system, and is the result of
Leukocyte esterase acting upon compatible esters. This ester/esterase
reaction produces a relatively unstable indoxyl moiety that is oxidized to
form an indigo color that is monitored by monochronatic spectrophotometry.
To enhance the speed, completeness and specificity of the indoxyl moiety
can be oxidized by the addition of a dehydrogenase to the reaction
solution that will oxidize (replace) the alcohol group on the indoxyl
group to yeild a ketone. This tranisitional indoxyl ketone radical formed
will enhance color development specificity, accuracy, and sensitivity of
the reaction. The reagent system may contain one or more of the following
compounds, 2,4-dinotrophenylhydrazine, hydroxylamine, or semicarbizide,
which in the presence of indoxide ketones will give color development that
can be monitored at the same wavelength as the indigo. A further
enhancement of the method concerning the indoxyl intermediate, is the
addition of p-dimethylaminobenzaldehyde or p-Nitrobenzenediazonium
tetrafluroborate (or other azo indicators), these will react with the
intermediate to enhance color development at the afore mentioned
wavelength. This reaction would enhance specificity, sensivitly, and
accuracy. The reagent is buffered depending on which group or single
component is used in the color developing reagent. The R2 if applicable
also contains a buffer to adjust sample pH and to aid in solubility and
compatibility of the R2's complex chemical matrix. This complex reagent
matrix requires a complementary aqueous buffering system with unique
dynamics capable of adjusting the reaction solution to the ideal pKa, and
promoting component solution compatibility with autoanalyzers. Unbuffered
solutions may have high acidic, or basic activity, or strictly organic
properties which are not compatible with autoanalyzer syringes, tubing,
metal, and plastic parts. This reagent system buffering is designed to
correct these problems. The R2 (if applicable) also contains surfactants
to decrease surface tension, promote effective mixing on a molecular
level, aid in carrier-free matrix, and improve flow dynamics through
tubing and syringes of automated analyzers. The preceding components and
concentration of components of R1 and/or R2 reagents can be varied to
compensate for limitations and variations in the configuration of sampling
and reagent delivery systems of various makes of autoanalyzers. Without
further elaboration, it is believed that one skilled in the art can, using
the preceding description, effectively utilize the present invention. The
following preferred specific embodiments are, therefore, to be construed
as merely illustrative, and not limitive of the remainder of the
disclosure of the present invention in any way whatsoever. In the
following examples, all instrument parameters, reagent combinations, and
method techniques are generalized.
EXAMPLE 1
The automated WBC urinalysis reagent system's single reagent system
contains surfactant, 2,3-Butanedione monoxime, dimercaptopropanol, bile
salts, albumin, calcium chloride, ethylenediametetraacetic acid, 3-indoyl
acetate, and buffer. The reagent is then placed in the autoanalyzer. The
urine sample, standards, and controls are placed on the autoanalyzer
specimen cups. The urine samples, standards, and controls are aliquoted
into cuvettes, mixed with the reagent, and read at specified intervals as
dictated by the instrument parameters at the specific wavelength
(monochromatically) depending on reagent combination used. In this
instance the assay should be read at 405 nanometers with read times
specific to the analyzer.
EXAMPLE 2
The automated WBC urinalysis reagent system's first reagent (R1) contains
surfactants, buffer, 2,3-Butanedione monoxime, ethylenediametetraacetic
acid, dimercaptopropanol, bile salts, albumin, calcium chloride, and
peroxidase. The second reagent (R2) consists of, some or all of the
following: hydrogen peroxide, 3-indolyl acetate, N-toluene sulfonyl
alanine indole ester, derivatized pyrrole amino acid ester, buffers,
and/or surfactants. The reagents are placed on the autoanalyzer. The urine
samples, standards, and controls are placed in the autoanalyzer specimen
cups. The urine samples, standards, and controls, are aliquoted into
cuvettes mixed with the first reagent, the second reagent is then added
and mixed, and the solutions are then read at specified intervals as
dictated by the instrument parameters at the specific wavelength
(monochromatically) depending on the reagent combination used. In this
instance the assay should be read at 405 nanometers with read time
specific to the analyzer.
EXAMPLE 3
The automated WBC urinalysis reagent system's first reagent (R1) contains
surfactants, buffer, bile salts, albumin, calcium chloride, 2,3-
Butanedione monoxime, dimercaptopropanol, and ethylenediametetraacetic
acid. The second reagent (R2) consists of some or all of the following:
dehydrogenase, 3-indolyl acetate, N-toluene sulfonyl alanine indole ester,
derivatized pyrrole amino acid ester, buffers, and/or surfactants. The
reagents are placed on the autoanalyzer. The urine samples, standards, and
controls are placed in the autoanalyzer specimen cups. The urine samples,
standards, and controls, are aliquoted into cuvettes mixed with the first
reagent, the second reagent is then added, and mixed, and the solutions
are then read at specified intervals as dictated by the instrument
parameters the specified wavelength (monochromatically) depending on the
reagent combination used. In this instance the assay should be read at 405
nanometers, with read times specific to the analyzer.
EXAMPLE 4
The automated WBC urinalysis reagent system's first reagent (R1) contains
sone or all of the following: surfactants, buffer, bile salts, albumin,
calcium chloride, 2,3- Butanedione monoxime, ethylenediametetraacetic
acid, dehydrogenase, 3-indolyl acetate, N-toluene sulfonyl alanine indole
ester, and derivatized pyrrole amino acid ester. The second reagent (R2)
consists of some or all of the following: 2,4-dinotrophenylhydrazine, 3-
indolyl acetate, N-toluene sulfonyl alanine indole ester, derivatized
pyrrole amino acid ester, buffers, and surfactants. The reagents are
placed on the autoanalyzer. The urine samples, standards, and controls are
placed in the autoanalyzer specimen cups. The urine samples, standards,
and controls, are aliquoted into cuvettes, mixed with the first reagent,
the second reagent is then added and mixed, and the solutions are then
read at specified intervals as dictated by the instrument parameters at
the specific wavelengths (monochromatically) depending on the reagent
combination used. In this instance the assay should be read at 405
nanometers and read times specific to the analyzer.
EXAMPLE 5
The automated WBC urinalysis reagent system's first reagent (R1) contains
some or all of the following: surfactants, buffer, bile salts, calcium
chloride, albumin, 2,3- Butanedione monoxime, ethylenediametetraacetic
acid, 3-indolyl acetate, N-toluene sulfonyl alanine indole ester, and/or
derivatized pyrrole amino acid ester, are added. The second reagent (R2)
consists of some or all of the following: p-dimethylaminobenzaldehyde,
3-indolyl acetate, N-toluene sulfonyl alanine indole ester, derivatized
pyrrole amino acid ester, buffers, dilute hydrochloric acid, and
surfactants. The reagents are placed on the autoanalyzer. The urine
samples, standards, and controls are placed in the autoanalyzer specimen
cups. The urine samples, standards, and controls, are aliquoted into
cuvettes, mixed with the first reagent, the second reagent is then added
and mixed, and the solutions are then read at specified intervals as
dictated by the instrument parameters at the specified wavelength
(monochromatically) depending on the reagent combination used. In this
instance the assay should be read at 405 nanometers, with read times
specific to the analyzer.
EXAMPLE 6
The automated WBC urinalysis reagent system's first reagent (R1) contains
some or all of the following: surfactants, buffer, 2,3- Butanedione
monoxime, ethylenediametetraacetic acid, bile salts, calcium chloride,
and/or albumin. The second reagent (R2) consists of some, or all of the
following: p-nitorphenyl stearate, phenyl laurate, buffers, and
surfactants. The reagents are placed on the autoanalyzer. The urine
samples, standards, and controls are placed in the autoanalyzer specimen
cups. The urine samples, standards, and controls, are aliquoted into
cuvettes, mixed with the first reagent, the second reagent is then added
and mixed, and the solutions are then read at specified intervals as
dictated by the instrument parameters at the specified wavelength
(monochromatically) depending on the reagent combination used. In this
instance, the assay should be read at 405 nanometers with read times
specific to the analyzer.
The automated urinalysis system reagents are individually designed for
optimum analysis of urinary components. The reagent system for pH of urine
is carrier-independent, and has specific agents added to compensate for
curve instability, and to improve accuracy, linearity, and precision. A
buffer is added to enhance this reagent's linearity. The buffer's
compositions, pH and pKa are dictated by the specific indicators included
in the formulation. The concentrations of reagent buffers and other
components can be varied to compensate for variations in the configuration
of sampling and reagent delivery systems of different makes of
autoanalyzers. The addition of buffers to compensate for urines with high
buffer capacities that will cause interference with the pH assay is an
obvious advancement over the previous art that had no primary buffer to
stabilize color development and promote carrier-free-independence. The
reagent also contains surfactants to enchance carrier-free matrix,
decrease surface tension, promote effective mixing on a molecular level,
and improve flow dynamics through tubing and syringes of automated
analyzers.
The reagent system for pH can consist of two reagents, an R1 (reagent one
of a two component system) and R2 (reagent two of a two component system),
or just a single reagent, an R1. The color developing component of the
reagent system is the water soluble indicators present in specific
spectrophotometrically compatible groups in an aqueous solution that is
compatible with autoanalyzers, components, and flow dynamics. These
indicators may include, but are not limited to, Bromcresol green, Thymol
Blue, Bromothymol Blue, Phenol red, Tropaeolin 000 no. 1, Alizarin yellow
GG, Bromphenol red, and Chlorophenol red all of which can monitored
spectrophotometrically. These indicators may be used singularly, or in any
combination thereof, but only in the water-soluble salt form. The R2 if
applicable, also contains a buffer to adjust sample pH and aid in
solubility and compatibility of the reagent's complex chemical matrix.
This complex chemical matrix requires a complimentary aqueous with unique
dynamics capable of adjusting the reaction solution to the ideal pKa, and
promoting reagent component solution compatibility with autoanalyzers.
Unbuffered solutions may have high acidic or basic activity, or strictly
organic properties which are not compatible with autoanalyzer syringes,
tubing, metal, and plastic parts. The buffer also promotes carrier
independence. The R2 (if applicable) also contains surfactants that allows
enhance the carrier-free matrix, decrease surface tension, promote
effective mixing on a molecular level, and improve flow dynamics through
tubing and syringes of automated analyzers. The concentrations of
components of the R1 and/or the R2 reagents can be varied to compensate
for limitations and variations in the configuration of sampling and
reagent delivery systems of various of makes of autoanalyzers. Without
further elaboration, it is believed that one skilled in the art can, using
the preceding description, effectively utilize the present invention to
its fullest extent. The following preferred specific embodiments are meant
to merely illustrate and not limit the remainder of the disclosure of this
present invention in any way whatsoever. In the following examples, all
automated instrument parameters, reagent combinations, and method
techniques are generalized.
EXAMPLE 1
The automated pH urinalysis reagent system's single reagent system contains
surfactant, buffers, Bromcresol green, Bromothymol blue, and Thymol Blue
(note: these three indicators are balanced quantatively and
compositionally to be in solution together to allow exact, and linear
spectrophotometric extrapolation of results for pH). The reagent is placed
on the autoanalyzer. The urine samples, standards, and controls are placed
in the autoanalyzer specimen cups. The urine samples, standards, and
controls, are aliquoted into cuvettes, mixed with the reagent, and read at
specified intervals as dictated by the instrument parameters, and at the
specified wavelength (monochromatically) depending on reagent combination
used. In this instance the assay should be read at 600 nanometers, and
read times are specific to the analyzer.
EXAMPLE 2
In the automated pH urinalysis single reagent system contains surfactants,
buffer, Alizarin yellow, Tropaeolin 000 no. 1, Cresol red, Phenol Red,
Bromphenol Red, Chlorophenol Red (note: these three indicators are
balanced and designed to be in solution together to allow exact and linear
spectrophotometric extrapolation of results for pH). The reagent is placed
on the autoanalyzer. The urine samples, standards, and controls are placed
in the autoanalyzer specimen cups. The urine sample, standards, and
control, are aliquoted into cuvettes, mixed with the reagent, and read at
specified intervals as dictated by the instrument parameters and at the
specified wavelength (monochromatically) depending on reagent combination
used. In this instance the assay should be read 405 nanometers and read
times are specific to the analyzer.
The automated urinalysis system reagents are individually designed for
their specific urinary component automated analysis in urine. The reagent
system for Specific Gravity in urine is carrier independent, and has
specific agents added to compensate for interference from, urinary
protein, highly buffered urines, abnormal pH and other normal urinary
constituents. The reagent system is composed of two reagents (but can
consist of one reagent). The first reagent (R1) is specifically designed
to neutralize matrix interference and increase sample-reagent
compatibility with the autoanalyzer. A buffer is added to the first
reagent (R1) to eliminate the affects of pH, highly buffered urines, and
other interfering substances (which cause increase buffer affects) by
nuetralizing pH. The buffer also aids in solubility and compatibility of
the complex chemical matrix. This complex chemical reagent/sample matrix
requires a complimentary buffering system with unique dynamics capable of
adjusting reaction soultion to the ideal pKa, and promoting component
solution compatibility with autoanalyzers. Unbuffered solutions may have
high amount of acidic and basic activity, or strictly organic properties
which are not compatible with autoanalyzer syringes, tubing, metal,
plastic parts), and the buffer promotes carrier independence. The R1 also
contains surfactants that enhance carrier free matrix, decrease surface
tension, promote effective mixing on a molecular level, and improve flow
dynamics through tubing and syringes of automated analyzers. Sodium
Thiosulfate is added to the R1 to enhance color developement through the
interaction which chloride present in urine (a major constituent). The R1
buffers constituents and concentrations can be varied in the to compensate
for limitations and variations in the configuration of sampling and
reagent delivery systems of various of makes of available autoanalyzers.
The reagent system for Specific Gravity second reagent (R2) is the color
generating reagent of the 2 reagent set (unless a single reagent system
for Specific Gravity is used). This second reagent R(2) is composed of
methyl vinyl ether copolymers (which are sensitive to ions in solution).
In the presence of ions in solution the vinyl group on the copolymer
reacts with ions in solution via an exchange reaction that yields a
hydrogen ion (H+). This exchange reactions effects a change in the pH of
the solution which is measured by the color change of an indicator or
combination of indictors including, but not limited to Thymol Blue,
Bromothymol Blue, and Litmus. One or more of these indicators can be used
in the R2. The advantage of using two or more indicators vs one would be
broadening the range of the color development. Isopropyl alcohol is added
to solubilize the copolymer. Please note that the prior art was restricted
to a carrier solid phase method because the polymers could not solubilize
to function independent of a carrier dependent solid matrix. The reagent
is buffered to a specific pH depending on the active group linked to the
vinyl copolymer and the corresponding indicators utilized for color
development. The R2 also aids in solubility and compatibility of the
reagents's complex chemical matrix. This complex chemical matrix requires
a complimentary, aqueous buffering system with unique fluid dynamics
capable of adjusting the reaction solution to the ideal pKa, promoting
reagent solution compatibility with autoanalyzers. Unbuffered solutions
may have acidic and basic activity, or strictly organic properties which
are not compatible with autoanalyzer syringes, tubing, metal, and plastic
parts, and promotes carrier independence. The R2 also contains surfactants
that enhance carrier free matrix, decrease surface tension, promote
effective mixing on a molecular level, and improve flow dynamics through
tubing, and syringes of automated analyzers. The components and
concentrations of components of R1 and/or the R2 reagents can be varied to
compensate for limitations, and configuration of sampling and reagent
delivery systems of various of makes of available autoanalyzers. Without
further elaboration, it is believed that one skilled in the art can, using
the preceding description, utilize the present invention to its fullest
extent. The following preferred specific embodiments are, therefore, to be
construed as merely illustrative, and not limit of the remainder of the
disclosure in anyway whatsoever. In the following examples, all instrument
parameters, reagent combinations, and method techniques are generalized.
EXAMPLE 1
In the automated urinalysis system reagents for Specific Gravity first
reagent (R1), contains surfactant, Buffer, and Sodium Thiosulfate. The
second reagent R2 consists of surfactant, buffer, methyl vinyl ether
copolymer, BromoThymol Blue, and Isopropyl Alcohol. The reagents are
placed on the autoanalyzer. The urine samples, standards, and controls are
placed in the autoanalyzer specimen cups. The urine samples, standards,
and controls, are aliquoted into cuvettes, mixed with the first reagent,
the second reagent is then added and mixed, and the solution is read at
specified intervals as dictated by the instrument parameters, and at the
specified wavelength (monochromatically) depending on reagent combination
used. In this instance, the assay should be read 660 nanometers, and read
times are specific to the analyzer.
EXAMPLE 2
In the automated urinalysis system reagents for Specific Gravity first
reagent (R1), contains surfactant, Buffer, and Sodium Thiosulfate. The
second reagent R2 consists of surfactant, buffer, methyl vinyl ether
copolymer, Bromothymol Blue, Thymol Blue and Isopropyl Alcohol. The
reagents are placed on the autoanalyzer. The urine samples, standards, and
controls are placed in the autoanalyzer specimen cups. The urine samples,
standards, and controls, are aliquoted into cuvettes, mixed with the first
reagent, the second reagent is then added and mixed, and the solution is
read at specified intervals as dictated by the instrument parameters, and
at the specified wavelength (monochromatically) depending on reagent
combination used. In this instance, the assay should be read 600
nanometers, and read times are specific to the analyzer.
The automated urinalysis system reagents are individually designed for
optimum analysis of specific urinary components automated analysis. The
reagent system for Total Ketone Bodies in urine is carrier independent,
and has specific agents added to compensate for interference from, enzyme
inhibitors, highly pigmented urines, sulfhydryl groups, aytipcal color
development, mesna (2-mercaptoethane sulfonic acid), levodopa, high ion
levels (specific gravity), abnormal pH and other normal urinary
constituents. The reagent system is composed of two reagents (but may
consist of one system, one reagent). The first reagent (R1) is
specifically designed to neutralize matrix interference and increase
sample-reagent compatibility, with the autoanalyzer. The component
2,3-Butanedione monoxime is included in this first reagent (R1) to remove
urea, and other substances found in urine that cause interference with the
colormetric reaction. Ethylenediaminetetraacetic acid and
dimercaptopropanol, are other components of the R1 that neutralize
interfering substances by chelation, remove enzyme inhibitors, and
anti-oxidant activity, These compounds removing oxidizing contaminants
such as hypochlorite, and act as a solution clarifyer. It causes the
disappearance of the characteristic yellow color of urine, thereby
enhancing spectrophotometric analysis. Bile salts (exp:cholic acid sodium
salt) are added to enhance solubility, enzyme activity, and prevent
denaturation of the enzyme. Delta-3 hydroxybutyrate dehydrogenase is added
to convert the B-hydroxybutyric acid (which composes 80% of Ketone Bodies
present in urine) to acetoacetic acid. The prior art does not address this
80% fraction of the ketone bodies in urine. B-Nicotinamide Adenine
Dinucleotide (NAD) is also included in the R1. The reaction of Delta-3
hydroxybutyrate dehydrogenase with the B-hydroxybutyric acid in the
presence of NAD, results in the reduction of the NAD to B-Nicotinamide
Adenine Dinucleotide (B-NADH). This reduction of NAD can be measured
spectrophotometrically at 340 nm, and corresponds directly to the quantity
of the B-Hydroxybutyric acid present. If desired the R1 as hereto-fore
described, can stand as a single reagent for determination of Ketone
Bodies. The total can be extrapolated from the B-Hydroxybutyric acid
fraction by multiplying its concentration by 1.25 (to compensate for the
20% fraction of Total Ketone Bodies due to acetoacetic acid). The R1 also
contains a buffer to adjust sample pH, establish carrier free matrix, aid
in solubility, and compatibility of the reagents's complex chemical
matrix. This complex chemical matrix requires a complementary aqueous
buffering system with unique dynamics capable of adjusting the reaction
solution to ideal pKa, and promoting reagent solution compatibility with
autoanalyzers. Unbuffered solutions have high acidic, or basic activity,
or strictly organic properties which are not compatible with autoanalyzer
syringes, tubing, metal, and plastic parts. The buffer also promotes
carrier independence. The R1 also contains surfactants that enhance the
carrier free matrix, decrease surface tension, promote effective mixing on
a molecular level, and improve flow dynamics through tubing, and syringes
of automated analyzers. The concentrations R1 buffers, and other
components can be varied to compensate for limitations, and variations in
the configuration of sampling and reagent delivery systems of various
makes of available autoanalyzers. The reagent buffers also compensate for
abnormal pH of urine samples, and urines with high buffer capacities. The
Total Ketone Bodies reagents system's second reagent (R2) is the color
generating reagent of the 2 reagent set (unless a single reagent system is
used). This second reagent is composed of Diazonium salts (e.g.,
4-Nitrobenzene diazonium tetrafluroborate) which couples with the
acetoacetic acid in the presence of sodium nitroferricyanide (or other
alkaline metal dyes), yielding a hydrazo compound that can be monitored at
645 nm. Note, the R1 component, D-3-Hydroxybutyrate dehydrogenase converts
B-Hydroxybutyric acid to acetoacetic acid. Thus, nearly all of the Ketone
Bodies in urine (99%) are in the form of acetoacetic acid. The remaining
1% is acetone. As a result, this method measures 99% of ketones bodies
compared to 20% measured by the prior art. The R2 also contains compounds
to enhance sodium nitroferricyanide stability and the ensuing color
development. These enhancers include (but are not limited to) alkali earth
compounds metals,: phosphoric acid trimorpholide (in an alkaline buffer),
ytrium (in an alkaline buffer), amine (or amine alcohols), and
Ethylenediaminetetraacetic acid. The reagent is buffered according to
which group, or single component is used in the color developing reaction.
The R2 also contains a buffer to adjust sample pH and aid in solubility,
and compatibility of the reagents complex chemical matrix. This complex
chemical matrix requires a complementary, aqueous buffering system with
unique dynamics capable of adjusting the reaction solution to the ideal
pKa, and promoting reagent solution compatibility autoanalyzers.
Unbuffered solutions may have high acidic and basic activity, or strictly
organic properties which are not compatible with autoanalyzer use of
syringes, tubing, metal, and plastic parts. The buffer also promotes
carrier independence. The R2 also contains surfactants that enhance the
carrier-free matrix, decrease surface tension, promote effective mixing on
a molecular level, and improve flow dynamics through tubing and syringes
of automated analyzers. The concentration and combination of components of
the R1 and/or the R2 reagents can be varied to compensate for limitations,
and variations in the configuration of sampling and reagent delivery
systems of various makes of available autoanalyzers. Without further
elaboration, it is believed that one skilled in the art can, using the
preceding description, utilize the present invention to its fullest
extent. The following preferred specific embodiments are, therefore, to be
construed as merely illustrative, and not limit of the remainder of the
disclosure in anyway whatsoever. In the following examples, all instrument
parameters, reagent combinations, and method techniques are generalized.
EXAMPLE 1
The automated Total Ketone Bodies urinalysis reagent system's first reagent
(R1) contains, surfactant, 2,3-Butanedione monoxime,
ethylenediametetraacetic acid (sodium salt), dimercaptopropanol, bile
salts, Delta-3-Hydroxybutyrate Dehydrogenase, NAD, and buffer. The second
reagent R2 consist of surfactant, buffer, 4-Nitrobenzene diazonium
tetrafluroborate, ethylenediametetraacetic acid (sodium salt), sodium
nitroferricyanide, Ytrium, and phosphoric acid trimorpholide. The reagents
are placed on the autoanalyzer. The urine samples, standards, and controls
are placed in the autoanalyzer specimen cups. The urine samples,
standards, and controls, are aliquoted into cuvettes, mixed with the first
reagent, the second reagent is added and mixed, and the solution is read
at specified intervals as dictated by the instrument parameters and at the
specified wavelength (monochromatically) depending on reagent combination
used. In this instance, the assay should be read 645 nanometers, and read
times are specific to the analyzer.
EXAMPLE 2
The automated Total Ketone Bodies urinalysis reagent system's single
reagent contains, 2,3-Butanedione monoxime, ethylenediametetraacetic acid,
bile salts, dimercaptopropanol, NAD, B-3-Hydroxybutyrate Hydrogenase,
buffers, and surfactants. The reagents are placed on the autoanalyzer. The
urine samples, standards, and controls are placed in the autoanalyzer
specimen cups. The urine samples, standards, and controls, are aliquoted
into cuvettes, mixed with the reagent, and the solution is read at
specified intervals as dictated by the instrument parameters and at the
specified wavelength monochromatically depending on reagent combination
used. In this instance the assay should be read at 340 nanometers
wavelength and read times are specific to the analyzer.
EXAMPLE 3
In the automated Total Ketone Bodies urinalysis reagent system's first
reagent (R1) contains surfactants, buffer, 2,3-Butanedione monoxime,
ethylenediametetraacetic acid, are added. The second reagent (R2) consists
of, buffer, 4-Nitrobenzene diazonium tetrafluroborate,
ethylenediametetraacetic acid (sodium salt), sodium nitroferricyanide,
Ytrium, and phosphoric acid trimorpholide. buffers, and surfactants. The
reagents are placed on the autoanalyzer. The urine samples, standards, and
controls are placed in the autoanalyzer specimen cups. The urine samples,
standards, and controls, are aliquoted into cuvettes, mixed with the first
reagent, the second reagent is added and mixed, and the solution is read
at specified intervals as dictated by the instrument parameters, and at
the specified wavelength (monochromatically) depending on reagent
combination used. In this instance the assay should be read 645 nanometers
and read times are is specific to the analyzer.
The automated urinalysis system reagents are individually designed for
optimum analysis of their specific urinary components. The reagent system
for Protein in urine is carrier independent and has specific agents added
to compensate for interference from, highly pigmented urines, enzyme
inhibitors, high ionic levels (specific gravity), abnormal pH (elevated),
quaternary ammonium compounds (i.e., from some antiseptics, and
detergents), or skin cleaners containing chlorhexidine, and other normal
urinary constituents. The reagent system is composed of two reagents (but
can consist of one reagent). The first reagent, (R1) is specifically
designed to neutralize matrix interference and increase sample-reagent
compatibility with the autoanalyzer. The compound 2,3-Butanedione monoxime
is included in this first reagent (R1) to remove urea, and other
substances found in urine that cause interference with the colormetric
reaction. Ethylenediaminetetraacetic acid, and dimercaptopropanol, are
other components of the R1 used to neutralize interfering substances by
chelation, neutralization of enzyme inhibitors, and anti-oxidant activity,
Thus by neutralizing contaminants such as hypochlorite. Also these
components act as a solution clarifyers (when added to urine it causes the
disappearance of the characteristic yellow color of urine, thus enhancing
spectrophotometric analysis). Potassium chloride and sodium chloride are
present to provide high ionic strength, which inturns increase
solubilization of proteins. Succinate buffer and citrate buffer are
present to optimize the pKa of the reagent system for analysis. The R1
also contains a buffer to aid in solubility and compatibility of multiple
chemicals that require a mutual buffering system with unique dynamics,
adjusting the reaction solution to the ideal pKa's, promotes reagent
solution compatibility with autoanalyzers. Unbuffered solutions may have
high acidic, or basic activity, or strictly organic properties which are
not compatible with autoanalyzer syringes, tubing, metal, and plastic
parts. The buffer also promotes carrier independence. The R1 also contains
surfactants that enhance the carrier- free matrix, decrease surface
tension, promote effective mixing on a molecular level, and improve flow
dynamics through tubing and syringes of automated analyzers. The
concentration of R1 buffers and other components can be varied to
compensate for limitations and variations in the configuration of sampling
and reagent delivery systems of various makes of autoanalyzers. The
buffers also compensate for abnormal pH of urine and urines samples and
urines with high buffer capacities.
The Protein reagent system's second reagent (R2) is the color generating
reagent of the 2 reagent set (unless a single reagent system for Protein
is used). This second reagent is composed of Copper sulfate in solution
with sodium hydroxide, potassium iodide, sodium and/or potassium tartrate,
and ARW-7 (wetting agent). The Cu++ ions bind with the unshared electrons
in the nitrogen and oxygen atoms of proteins to form a blue-violet complex
which can be measured spectrophotometrically at 540 nm. Bromcresol green
exhibits a measurable dye-binding complex in the presence of albumin.
Other indicators present are Coomassie Blue, tetrabromphenol blue, and
2,2'-biquinoline-4,4'-dicarboxylic acid disodium salt dihydrate
(intensifies color development, thereby increasing sensitivity). The
reagent is buffered depending on which group or single component is used
in the color developing reaction. The R2 also contains a buffer to adjust
sample pH, aid in solubility, and compatibility of these reagent's complex
chemical matrix. This complex chemical matrix requires a complementary
aqueous buffering system with unique dynamics capable of adjusting the
reaction solution to the ideal pKa, and promoting reagent component
solution compatibility with autoanalyzers. Unbuffered solutions may have
high acidic, or basic activity, or strictly organic properties which are
not compatible with autoanalyzer syringes, tubing, metal, and plastic
parts. The buffering systems also promotes carrier independence. The R2
also contains surfactants that enhance the carrier-free matrix, decrease
surface tension, promote effective mixing on a molecular level, and
improve flow dynamics through tubing and syringes of automated analyzers.
The concentrations and combinations of components of the R1 and/or the R2
reagents can be varied to compensate for limitations, and variations in
the configuration of sampling and reagent delivery systems of various of
makes of autoanalyzers. Without further elaboration, it is believed that
one skilled in the art can, using the preceding description, effectively
utilize the present invention. The following preferred specific
embodiments are meant to merely illustrate, and not limit the remainder of
the disclosure of the present invention in any way whatsoever. In the
following examples, all instrument parameters, reagent combinations, and
method techniques are generalized.
EXAMPLE 1
The automated Protein urinalysis reagents system's first reagent (R1)
contains surfactant, 2,3-Butanedione monoxime, ethylenediametetraacetic
acid (sodium salt), dimercaptopropanol, potassium chloride, sodium
chloride, and buffer. The second reagent R2 consists of surfactant,
buffer, copper sulfate, sodium hydroxide, potassium iodide, sodium and/or
potassium tartrate, ARW-7, and 2,2'-biquinoline-4,4'-dicarboxylic acid
disodium salt dihydrate. The reagents are placed on the autoanalyzer. The
urine samples, standards, and controls are placed in the autoanalyzer
specimen cups. The urine samples, standards, and controls are aliquoted
into cuvettes, mixed with the first reagent, the second reagent is then
added and mixed, and the solution is read at specified intervals as
dictated by the instrument parameters at the specified wavelength
(monochromatically) depending on the reagent combination used. In this
instance the assay should be read 540 nanometers, with read times specific
to the analyzer.
EXAMPLE 2
The automated Protein urinalysis reagent system's single reagent system
contains 2,3-Butanedione monoxime, ethylenediametetraacetic acid,
potassium chloride, sodium chloride, dimercaptopropanol, copper sulfate,
sodium hydroxide, potassium iodide, sodium and/or potassium tartrate,
2,2'- biquinoline-4,4'-dicarboxylic acid disodium salt, buffers, and
surfactants. The reagents are placed on the autoanalyzer. The urine
samples, standards, and controls are placed in the autoanalyzer specimen
cups. The urine samples, standards, and controls are aliquoted into
cuvettes, mixed with the reagent, and the solution is read at specified
intervals as dictated by the instrument parameters, and at the specified
wavelength (monochromatically) depending on reagent combination used. In
this instance the assay should be read at 540 nanometers wavelength, with
read times specific to the analyzer.
EXAMPLE 3
The automated Protein urinalysis reagent system's first reagent (R1)
contains surfactants, buffer, 2,3-Butanedione monoxime,
ethylenediametetraacetic acid, dimercaptopropanol, succinate buffer, and
bromcresol green. The reagents are placed on the autoanalyzer. The urine
samples, standards, and controls are placed in the autoanalyzer specimen
cups. The urine samples, standards, and controls are mixed with the first
reagent, the second reagent then is aliquoted into cuvettes, added and
mixed, and the solution is read at specified intervals as dictated by the
instrument parameters, and at the specified wavelength bichromatically
depending on reagent combination used. In this instance, the assay should
be read at 660, and 750 nanometers, and read times are specific to the
analyzer.
EXAMPLE 4
The automated Protein urinalysis reagent system's first reagent (R1)
contains surfactant, 2,3-Butanedione monoxime, ethylenediametetraacetic
acid (sodium salt), dimercaptopropanol, potassium chloride, sodium
chloride, and buffer. The second reagent R2 consists of surfactant,
succinate buffer, and bromcresol green. The reagents are placed on the
autoanalyzer. The urine samples, standards, and controls are placed in the
autoanalyzer specimen cups. The urine samples, standards, and controls are
aliquoted into cuvettes, mixed with the first reagent, the second reagent
is then added, and the solution is read at specified intervals as dictated
by the instrument parameters, and at the specified wavelength
(monochromatically) depending on reagent combination used. This assay
should be read at 540 nanometers, and read times are is specific to the
analyzer.
EXAMPLE 5
The automated Protein urinalysis reagent system's, first reagent (R1)
contains surfactant, 2,3-Butanedione monoxime, ethylenediametetraacetic
acid (sodium salt), dimercaptopropanol, potassium chloride, sodium
chloride, and buffer. The second reagent, R2 consists of buffer,
surfactant, and tetrabromphenol blue. The reagents are placed on the
autoanalyzer. The urine samples, standards, and controls are placed in the
autoanalyzer specimen cups. The urine samples, standards, and controls are
aliquoted into cuvettes, mixed with the first reagent, the second reagent
is then added and mixed, and the solution is read at specified intervals
as dictated by the instrument parameters and at the specified wavelength
(monochromatically) depending on reagent combination used. In this
instance, the assay should be read at 600 nanometers, and read times are
is specific to the analyzer.
EXAMPLE 6
The automated Protein urinalysis reagent system's the first reagent, (R1)
contains surfactant, 2,3-Butanedione monoxime, ethylenediametetraacetic
acid (sodium salt), dimercaptopropanol, potassium chloride, sodium
chloride, and buffer. The second reagent, (R2) consists of buffer,
surfactant, Coomassie blue, alcohol, and tetrabromphenol blue. The
reagents are placed on the autoanalyzer. The urine samples, standards, and
controls are placed in the autoanalyzer specimen cups. The urine samples,
standards, and controls are aliquoted into cuvettes, mixed with the first
reagent, the second reagent is then added and mixed, and the solution is
read at specified intervals as dictated by the instrument parameters, and
at the specific wavelength (monochromatically) depending on the reagent
combination used. In this instance, the assay should be read at 600
nanometers, and read times are specific to the analyzer.
The automated Glucose urinalysis reagent system's are individually designed
for optimum analysis of their specific urinary components. The reagent
system for Glucose in urine is carrier independent, and has specific
agents added to compensate for interference from enzyme inhibitors, Ketone
Bodies, high ionic strength urine samples (specific gravity), Vitamin C,
and other abnormal amounts urinary constituents. The reagent system is
composed of two reagents (but can consist of one reagent). The first
reagent (R1), is specifically designed to neutralize matrix interference
and increase sample-reagent compatibility. The compound, 2,3-Butanedione
monoxime, is included in this first reagent, (R1) to remove urea and other
substances found in urine that cause interference with the colormetric
reaction. Ethylenediaminetetraacetic acid and dimercaptopropanol are other
components of the R1 that neutralize interfering substances by chelation,
neutralize enzyme inhibitors, and anti-oxidant activity. These compounds
remove oxidizing contaminants such as hypochlorite, and act as a solution
clarifyers. They remove the characteristic yellow color of urine, thereby
enhancing spectrophotometric analysis. This reagent may also contain
Glucose oxidase which converts urinary glucose to gluconic acid. During
oxidation, hydrogen peroxide is formed as a side product. Adenosine
triphosphate (ATP) when added, in the presence of hexokinase will convert
glucose to glucose-6-phosphate. Both of these compounds may be included in
this reagent. The R1 also contains a buffer to adjust sample pH, and aid
in solubility and compatibility of the reagent's complex chemical matrix.
This complex matrix requires a complementary, aqueous buffering system
with unique dynamics capable of adjusting the reaction solution to the
ideal pKa and promoting the reagent component solution compatibility with
autoanalyzers. Unbuffered solutions may have high of acidic or basic
activity, or strictly organic properties which are not compatible with
autoanalyzer syringes, tubing, metal, and plastic parts. The buffer also
promotes carrier independence. The R1 also contains surfactants that
enhance the carrier-free matrix, decrease surface tension, promote
effective mixing on a molecular level, and improve flow dynamics through
tubing and syringes of automated analyzers. The concentrations of R1
buffers and other components can be varied to compensate for limitations
and variations in the configuration of sampling, and reagent delivery
systems of various makes of autoanalyzers. The buffers also compensate for
abnormal pH of urine samples and urines with high buffer capacities.
The Glucose reagent system's second reagent (R2), is the color generating
reagent of the 2 reagent set (unless a single reagent system for Glucose
is used). This second reagent is composed of one or more of the following:
Peroxidase (which converts or oxidizes the newly formed hydrogen peroxide
product of the glucose oxidase reaction, and releases an oxygen),
o-Dianisidine, ampyrone, phenol, p-hydroxybenzoic acid, potassium iodide
chromogen, and N-Ethyl-N-(2-hydroxy-3-sulopropyl)-m-toluidine. The latter
7 can be used singularly, or in groups as couplers with Ampyrone (4-AA).
The 4-AA is reduced by the oxygen released from the hydrogen
peroxide/peroxidase reaction. Glucose-6-phosphate dehydrogenase is added
to oxidize the glucose-6-phosphate present from the hexokinase reaction.
NADP+ and/or NAD+ are added to acy as hydrogen acceptors from the
glucose-6-phosphate, or the glucose dehydrogenase reaction. Note, glucose
dehydrogenase is added to oxidize glucose to d-glucono-gamma-lactone. When
this occurs NAD+ is reduced to NADH and can be monitored
spectrophotometrically at 340 nm. The buffer is added to adjust sample pH,
aid in solubility, and compatibility of the reagent complex chemical
matrix. This complex chemical matrix requires a complementary, aqueous
buffering system with unique dynamics capable of adjusting the reaction
solution to the ideal pKa, and promoting the reagent component solution
compatibility with autoanalyzers. Unbuffered solutions may have high
acidic or basic activity, or strictly organic properties which are not
compatible with autoanalyzer syringes, tubing, metal, and plastic parts.
This buffer also promotes carrier independence. The R2 also contains
surfactants that enhance the carrier-free matrix, decrease surface
tension, promote effective mixing on a molecular level, and improve flow
dynamics through tubing, and syringes of automated analyzers. The
concentration and combination of components of the R1 and/or the R2
reagents can be varied to compensate for limitations, and variations in
the configuration of sampling, and reagent delivery systems of various
makes of autoanalyzers. Without further elaboration, it is believed that
one skilled in the art can, using the preceding description, effectively
utilize the present invention. The following preferred specific
embodiments are meant to merely illustrate, and not limit the remainder of
the disclosure of the present invention in any way whatsoever. In the
following examples, all instrument parameters, reagent combinations, and
method techniques are generalized.
EXAMPLE 1
The automated Glucose urinalysis reagent system's first reagent (R1),
contains surfactant, 2,3-Butanedione monoxime, ethylenediametetraacetic
acid (sodium salt), dimercaptopropanol, and buffer. The second reagent
(R2), consists of surfactant, buffer, glucose oxidase, 4-AA, EHSPT (one or
more of the following maybe substituted for: o-Dianisidine, ampyrone,
phenol, p-hydroxybenzoic acid, potassium iodide chromogen, or
N-Ethyl-N-(2-hydroxy-3-sulopropyl)-m-toluidine). The reagents are placed
on the autoanalyzer. The urine samples, standards, and controls are placed
in the autoanalyzer specimen cups. The urine samples, standards, and
controls are aliquoted into cuvettes, mixed with the first reagent, the
second reagent is then added and mixed, and the solution is read at
specified intervals as dictated by the instrument parameters at the
specified wavelength (monochromatically) depending on reagent combination
used. In this instance the assay should be read at 555 nanometers, and
read times are specific to the analyzer.
EXAMPLE 2
The automated Glucose urinalysis reagent system's first reagent (R1),
contains 2,3-Butanedione monoxime, ethylenediaminetetraacetic acid,
dimercaptopropanol, buffers, Glucose oxidase, and surfactants. The second
reagent (R2), contains 4-AA, EHSPT (and one or more of the following:
o-Dianisidine, ampyrone, phenol, p-hydroxybenzoic acid, potassium iodide
chromogen, and N-Ethyl-N-(2-hydroxy-3-sulopropyl)-m-toluidine). The
reagents are placed on the autoanalyzer. The urine samples, standards, and
controls are placed in the autoanalyzer specimen cups. The urine samples,
standards, and controls are aliquoted into cuvettes, mixed with the
reagent's R1 and R2, and the solution is read at specified intervals as
dictated by the instrument parameters at the specified wavelength
(monochromatically) depending on reagent combination used. In this
instance, the assay should be read at 555 nanometers and read times are
specific to the analyzer.
EXAMPLE 3
The automated Glucose urinalysis reagent system's first reagent (R1),
contains surfactants, buffer, 2,3-Butanedione monoxime, Glucose oxidase,
ethylenediaminetetraacetic acid, dimercaptopropanol, and NAD+. In the
second reagent (R2), contains glucose dehydrogenase, buffers, and
surfactants. The reagents are placed on the autoanalyzer. The urine
samples, standards, and controls are placed in the autoanalyzer specimen
cups. The urine samples, standards, and controls are aliquoted into
cuvettes, mixed with the first reagent, the second reagent is then added
and mixed, and the solution is read at specified intervals as dictated by
the instrument parameters and at the specified wavelength
(monochromatically) depending on reagent combination used. In this
instance the assay should be read at 340 nanometers, and read times are
specific to the analyzer.
EXAMPLE 4
In the automated Glucose urinalysis reagent system's single reagent system
(R1), contains surfactant, NAD+ or NADP+, 2,3-Butanedione monoxime,
ethylenediametetraacetic acid, dimercaptopropanol, buffers, and Glucose
oxidase. The reagents are placed on the autoanalyzer. The urine samples,
standards, and controls are placed in the autoanalyzer specimen cups. The
urine samples, standards, and controls are aliquoted into cuvettes, mixed
with the reagent, and the solution is read at specified intervals as
dictated by the instrument parameters, and at the specified wavelength
(monochromatically) depending on reagent combination used. In this
instance, the assay should be read at 340 nanometers, and read times are
specific to the analyzer.
EXAMPLE 5
The automated Glucose urinalysis reagent system's first reagent (R1),
contains surfactant, 2,3-Butanedione monoxime, ethylenediametetraacetic
acid (sodium salt), dimercaptopropanol, and buffer. The second reagent R2,
contains ATP, Hexokinase, Glucose-6-phosphate dehydrogenase, NADP+ and/or
NAD+, buffer, and surfactant. The reagents are placed on the autoanalyzer.
The urine samples, standards, and controls are placed in the autoanalyzer
specimen cups. The urine samples, standards, and controls are aliquoted
into cuvettes, mixed with the first reagent, the second reagent is then
added and mixed, and the solution is read at specified intervals as
dictated by the instrument parameters, and at the specified wavelength
(monochromatically) depending on reagent combination used. In this
instance, the assay should be read at 340 nanometers, and read times are
specific to the analyzer.
EXAMPLE 6
The automated glucose urinalysis reagent system's first reagent (R1),
contains surfactant, 2,3-Butanedione monoxime, ethylenediaminetetraacetic
acid (sodium salt), dimercaptopropanol, ATP, Hexokinase, and buffer. The
second reagent (R2), contains Glucose-6-phosphate dehydrogenase, NADP+
and/or NAD+, buffer, and surfactant. The reagents are placed in the
autoanalyzer. The urine samples, standards, and controls are placed on the
autoanalyzer specimen cups. The urine samples, standards, and controls are
aliquoted into cuvettes, mixed with the first reagent, the second reagent
is the added and mixed, and the solution is read at specified intervals as
dictated by the instrument parameters, and at the specified wavelength
(monochromatically) depending on reagent combination used. In this
instance, the assay should be read at 340 nanometers, and read times are
specific to the analyzer.
EXAMPLE 7
The automated Glucose urinalysis reagent system's first reagent (R1),
contains surfactant, 2,3-Butanedione monoxime, ethylenediaminetetraacetic
acid (sodium salt), dimercaptopropanol, and buffer. The second reagent R2,
contains Glucose-6-phosphate dehydrogenase, NADP+ and/or NAD+, buffer,
Glucose oxidase, Hexokinase, and surfactant. The reagents are placed on
the autoanalyzer. The urine samples, standards, and controls are placed in
the autoanalyzer specimen cups. The urine samples, standards, and controls
are aliquoted into cuvettes, mixed with the first reagent, the second
reagent is then added and mixed, and the solution is read at specified
intervals as dictated by the instrument parameters, and at the specified
wavelength (monochromatically) depending on reagent combination used. In
this instance, the assay should be read at 340 nanometers, and read times
are specific to the analyzer.
EXAMPLE 8
The automated Glucose urinalysis reagent system's single reagent (R1),
system contains surfactant, 2,3-Butanedione monoxime,
ethylenediametetraacetic acid (sodium salt), dimercaptopropanol, buffer,
Glucose-6-phosphate dehydrogenase, NADP+ and/or NAD+, buffer, Glucose
oxidase, and Hexokinase. The reagents are placed on the autoanalyzer. The
urine samples, standards, and controls are placed in the autoanalyzer
specimen cups. The urine samples, standards, and controls are aliquoted
into cuvettes, mixed with the first reagent, the second reagent is the
added and mixed, and the solution is read at specified intervals as
dictated by the instrument parameters, and at the specified wavelength
(monochromatically) depending on reagent combination used. In this
instance, the assay should be read at 340 nanometers, and read times are
specific to the analyzer.
The automated urinalysis system reagents are individually designed for
optimum analysis of specific urinary components. The reagent system for
Bacterial Reductase/Nitrite/Indole activity (as a measure for bacterial
uremia) in urine is carrier independent, and has specific agents added to
compensate for interference from enzyme inhibitors, and other abnormal
amounts of urinary constituents. The reagent system is composed of two
reagents, but can be consist of one reagent. The first reagent (R1), is
specifically designed to neutralize matrix interference, and increase
sample to liquid reagent compatibility with the autoanalyzer. The
component, 2,3-Butanedione monoxime, is included in this first reagent
(R1) to remove urea, and other substances found in urine that cause
interference with the colormetric reaction. Ethylenediaminetetraacetic
acid, and dimercaptopropanol are other components of the R1 that
neutralize interfering substances by chelation, inactivation of enzyme
inhibitors, and anti-oxidant activity. These compounds remove oxidizing
contaminants such as hypochlorite, and act as solution clarifyers (i.e.,
they absorb or cause the disappearance of the characteristic yellow color
of urine), thereby enhancing spectrophotometric analysis. Oxidized
Glutathione (GSSG) in one of several analytical pathways is present to act
as a substrate for the bacterial reductase. B-Nicotinamide Adenine
Dinucleotide Phosphate (reduced form, NADPH), and/or Nicotinamide Adenine
Dinucleotide (reduced form, NADH) are present to act as coenzymes for the
reductase enzyme reaction. Utilizing another analytical pathway the R1
would would contain the above referenced components to neutralize sample
matrix interference and one or more of the following: Sulfuric acid,
Phosphoric acid, p-Arsanilic acid, Sulfanilamide,
N-Sulfanilylsulfanilamide, and/or sodium iodide (or other salt forms). The
R1 also contains a buffer to adjust sample pH, aid in solubility and
compatibility of the reagent's complex chemical matrix. This complex
chemical matrix requires a complementary aqueous buffering system with
unique dynamics capable of adjusting reaction solution to the ideal pKa
and promoting reagent component solution compatibility with autoanalyzers.
Unbuffered solutions may have high acidic or basic activity, or strictly
organic properties which are not compatible with autoanalyzer syringes,
tubing, metal, and plastic parts. The buffer also promotes carrier
independence. The R1 also contains surfactants that enhance the carrier
free matrix, decrease surface tension, promote effective mixing on a
molecular level, and improve flow dynamics through tubing and syringes of
automated analyzers. The R1 buffers constituents and concentrations can be
varied to compensate for variations in the configuration of sampling, and
reagent delivery systems of various makes of autoanalyzers. The buffers
also compensate for abnormal pH of urine and urines with high buffering
capacities.
The Bacterial Reductase/Nitrite/Indole reagent system's second reagent (R2)
is the color generating reagent of the 2 reagent set unless a single
reagent system is used. This second reagent (R2) may utilize a reaction
pathway that requires one or more of the following: GSSG, NADPH, and NADH.
p-Dimethy-aminobenzaldehyde (DMABA) is an indicator for aerobic and
anaerobic activity correlated to indole production. Utilizing another
analytical pathway the R2 would contain one or more of the following: a
salt of iodide (Na, K, etc. . . . ), N-(1-napthyl)ethylenediamine,
1,2,3,4,-Tetrahydroisoquinoline hydrochloric acid, 4-Nitrobenzenediazonium
tetrafluroborate, or another suitable azo dye that forms a complex with
the diazonium salt, which can be measured spectrophotometrically at 540
nm. This second reagent (R2) may utilize a reaction pathway that requires
one or more of the following: Triphenyltetrazolium chloride act as a
substrate for the bacterial reductase, and when reduced yields a
colormetrically measurable compound. In the presence of the NADH and/or
NADPH reduced triphenyltetrazolium chloride will also yield a color
reaction at 340 nanometers. The buffers are added to adjust sample pH, aid
in solubility, and compatibility of the reagent's complex chemical matrix.
This complex chemical matrix requires a complementary aqueous buffering
system with unique dynamics capable of adjusting the reaction solution to
the ideal pKa, and promoting reagent component solution compatibility with
autoanalyzers. Unbuffered solutions may have high acidic and basic
activity, or strictly organic properties which are not compatible with
autoanalyzer syringes, tubing, metal, and plastic parts. The buffers also
promote carrier independence. The R2 also contains surfactants that
enhance the carrier free matrix, decrease surface tension, promote
effective mixing on a molecular level, and improve flow dynamics through
tubing and syringes of automated analyzers. The preceding components and
the concentrations of the components of the R1 and/or the R2 reagents can
be varied to compensate for limitations, variations in the configuration
of sampling, and reagent delivery systems of various of makes of
autoanalyzers. The above constituents can be varied, to compensate for
said differences. Without further elaboration, it is believed that one
skilled in the art can, using the preceding description, can effectively
utilize the present invention. The following preferred specific
embodiments are, therefore, to be construed as merely illustrative, and
not limitive of the remainder of the disclosure in anyway whatsoever. In
the following examples, all instrument parameters, reagent combinations,
and method techniques are generalized.
EXAMPLE 1
In the automated urinalysis system reagents for Bacterial reductase assay
in the first reagent (R1), contains surfactant, 2,3-Butanedione monoxime,
ethylenediaminetetraacetic acid (sodium salt), dimercaptopropanol, buffer.
The second reagent R2 consist of surfactant, buffer, GSSH, NADPH and or
NADH. The reagents are placed in the autoanalyzer. The urine sample,
standards, and controls are placed in the autoanalyzer specimen cups. The
urine sample, standards, and control, are mixed with the first reagent,
then the second reagent is added, and the solution is mixed, and read at
specified intervals as dictated by the instrument parameters, and at the
specified wavelength (monochromatically) depending on reagent combination
used. In this instance the assay should be read 340 nanometers and read
times are specific to the analyzer.
EXAMPLE 2
In the automated urinalysis system reagent for Bacterial reductase in the
dual reagent system, 2,3-Butanedione monoxime, ethylenediaminetetraacetic
acid, dimercaptopropanol, buffers, sulfanilamide, phosphoric acid (or
another suitable acid), surfactants, are added. In the R2
N-(1-naphthyl)ethylenediamine and or 1,2,3,4,-Tetrahydroisoquinoline
hydrochloric acid, or 1,2,3,4-tetrahydrobenzoquinolin-3-ol (or other
suitable azo dye), The reagents are placed in the autoanalyzer. The urine
sample, standards, and controls are placed in the autoanalyzer specimen
cups. The urine sample, standards, and control, are mixed with the
reagent, and the solution is read at specified intervals as dictated by
the instrument parameters and at the specified wavelength
monochromatically depending on reagent combination used. In this instance
the assay should be read at 540 nanometers wavelength and read times are
specific to the analyzer.
EXAMPLE 3
In the automated urinalysis system reagents for Bacterial reductase, the
first reagent (R1), contains surfactants, buffer, 2,3-Butanedione
monoxime, Glucose oxidase, ethylenediaminetetraacetic acid, and
dimercaptopropanol. In the R2 (second reagent) p-arsanilic acid,
1,2,3,4-tetrahydrobenzoquinolin-3-ol, buffers, and surfactants are added.
The reagents are placed in the autoanalyzer. The urine samples, standards,
and controls are placed in the autoanalyzer specimen cups. The urine
samples, standards, and controls, are mixed with the first reagent, then
the second reagent is added, and the solution is mixed, read at specified
intervals as dictated by the instrument parameters, and at the specified
wavelength monochromatically depending on reagent combination used. In
this instance the assay should be read 540 nanometers and read times are
specific to the analyzer.
EXAMPLE 4
In the automated urinalysis system reagents for Bacterial reductase in the
single reagent system (R1), contains surfactant, NADH and or NADPH,
2,3-Butanedione monoxime, ethylenediaminetetraacetic acid,
dimercaptopropanol, buffers, GSSH. The reagents are placed in the
autoanalyzer. The urine samples, standards, and controls are placed in the
autoanalyzer specimen cups. The urine samples, standards, and controls,
are mixed with the first reagent, then the second reagent is added, and
the solution is mixed, and read at specified intervals as dictated by the
instrument parameters at the specified wavelength (monochromatically)
depending on reagent combination used. In this instance the assay should
be read 340 nanometers and read times are specific to the analyzer.
EXAMPLE 5
In the automated urinalysis system reagents for Bacterial reductase, the
first reagent (R1), contains surfactant, 2,3-Butanedione monoxime,
ethylenediaminetetraacetic acid (sodium salt), dimercaptopropanol,
sulfuric acid, and buffer. The second reagent R2 consist of potassium
iodide, starch, buffer, and surfactant. The reagents are placed in the
autoanalyzer. The urine samples, standards, and controls are placed in the
autoanalyzer specimen cups. The urine samples, standards, and control, are
mixed with the first reagent, then the second reagent is added, and the
solution is mixed, and read at specified intervals as dictated by the
instrument parameters, and at the specified wavelength (monochromatically)
depending on reagent combination used. In this instance the assay should
be read 600 nanometers and read times are specific to the analyzer.
EXAMPLE 6
In the automated urinalysis system reagents for Bacterial reductase first
reagent (R1), contains surfactant, 2,3-Butanedione monoxime,
ethylenediaminetetraacetic acid (sodium salt), dimercaptopropanol, and
buffer. The second reagent R2 consist of Triphenyltetrazolium chloride,
NADPH and or NADH, buffer, and surfactant. The reagents are placed on the
autoanalyzer. The urine samples, standards, and controls are placed in the
autoanalyzer specimen cups. The urine samples, standards, and controls are
mixed with the first reagent, then the second reagent is added, and the
solution is mixed, and read at specified intervals as dictated by the
instrument parameters, and at the specified wavelength (monochromatically)
depending on reagent combination used. In this instance the assay should
be read 340 nanometers and read times are is specific to the analyzer.
EXAMPLE 7
In the automated urinalysis system reagents for Bacterial reductase assay
in the first reagent (R1), contains surfactant, 2,3-Butanedione monoxime,
ethylenediaminetetraacec acid (sodium salt), dimercaptopropanol, buffer.
The second reagent R2 consist of surfactant, buffer,
p-Dimethylaminobenzaldehyde (DMABA). The reagents are placed in the
autoanalyzer. The urine sample, standards, and controls are placed in the
autoanalyzer specimen cups. The urine samples, standards, and control are
mixed with the first reagent, then the second reagent is added, and the
solution is mixed, and read at specified intervals as dictated by the
instrument parameters, and at the specified wavelength (monochromatically)
depending on reagent combination used. In this instance the assay should
be read 540 nanometers and read times are specific to the analyzer.
From the foregoing it is believed that those familiar with the art will
readily recognize and appreciate the novel concepts and features of the
present invention. Numerous variations, changes and substitutions of
equivalents will present themselves from persons skilled in the art and
may be made without necessarily departing from the scope and principles of
this invention. Therefore the invention has been described with reference
to a number of its embodiment, it can nevertheless be arbitrarily varied
within the scope of the following claims.
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